Bispecific intracellular delivery vehicles

ABSTRACT

A composition for delivering an agent to a cell, comprising a bispecific affinity reagent and a pH-responsive, membrane destabilizing polymer. The bispecific affinity reagent may include a first affinity reagent covalently linked to a second affinity reagent, wherein the first affinity reagent binds to a molecule on the surface of a cell, and the second affinity reagent binds to an intracellular target.

This application is the National Stage of International Application No.PCT/US2009/043852, filed May 13, 2009, which claims the benefit ofProvisional Application Nos. 61/171,381, filed Apr. 21, 2009,61/140,779, filed Dec. 24, 2008, 61/140,774, filed Dec. 24, 2008,61/112,054, filed Nov. 6, 2008, and 61/112,048, filed Nov. 6, 2008. Eachapplication is incorporated herein by reference in its entirety.

STATEMENT OF JOINT RESEARCH AGREEMENT

The subject matter of the claimed invention was made as a result ofactivities undertaken within the scope of a joint research agreement,within the meaning of 35 U.S.C. §103(c)(3) and 37 C.F.R.§1.104(c)(4)(ii), by or on behalf of the University of Washington andPhaseRx, Inc., that was in effect on or before the claimed invention wasmade.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The invention was made with Government support under Contract No.2R01EB002991-05A1, awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention is in the field of the intracellular delivery oftherapeutic agents, and more particularly in the area of enhancement oftransport or delivery of molecules into the cell cytosol, usingbispecific affinity reagents and pH-responsive, membrane destabilizingpolymers.

It is often difficult to deliver compounds, such as proteins, geneticmaterial, and other drugs and diagnostic compounds, intracellularlybecause cell membranes resist the passage of these compounds. Variousmethods have been developed to administer agents intracellularly. Forexample, genetic material has been administered into cells in vivo, invitro and ex vivo using viral vectors, DNA/lipid complexes andliposomes. DNA has also been delivered by synthetic cationic polymersand copolymers and natural cationic carriers such as chitosan. Sometimesthe synthetic polymers are hydrophobically modified to enhanceendocytosis. While viral vectors are efficient, questions remainregarding the safety of a live vector and the development of an immuneresponse following repeated administration. Lipid complexes andliposomes appear less effective at transfecting DNA into the nucleus ofthe cell and may potentially be destroyed by macrophages in vivo.

Receptor mediated endocytosis offers an alternative means to targetspecific cell types and to deliver therapeutic agents intracellularly.Receptor-mediated endocytosis (RME) occurs when ligands bind to cellsurface receptors on eukaryotic cell membranes, initiating oraccompanying a cascade of phenomena culminating in the cellularinvagination of membrane complexes within clathrin-coated vesicles.Compounds which interact with specific cell surface receptors areemployed to target specific cell surface receptors. The compounds areendocytosed into the endosomes once the compounds interact with the cellsurface receptors. Linkages have been made directly with the compounds,or, in the case of DNA, through conjugation with polycationic polymerssuch as polylysine and DEAE-dextran which are then complexed with theDNA. (Haensler et al., Bioconj. Chem., 4:372-379 (1993)).

Even after therapeutic agents are delivered intracellularly, normaltrafficking in the cell can minimize their effectiveness. For example,certain antibody-antigen conjugates are readily endocytosed. However,after endocytosis, the antibody is not released into the cytosol butrather remains isolated in endosomes until it is trafficked to alysosome for degradation. (Press, O. W. et al., Cancer Research, 48:2249-2257 (1988)). Endosomes are membrane bound phospholipid vesicleswhich function in intracellular trafficking and degradation ofinternalized proteins. The internal pH of the endosomes is between 5.0and 5.5. Genetic material, being negatively charged, is often complexedwith polycationic materials, such as chitosan and polylysine, fordelivery to a cell. Both immunotherapy and gene therapy usingpolycation/nucleic acid complexes are limited by trafficking of thecomplexes by the cell from endosomes to lysosomes, where the antibodyconjugates or nucleic acids are degraded and rendered ineffective.

Protein transduction domains (PTDs) have attracted considerable interestin the drug delivery field for their ability to translocate acrossbiological membranes. The PTDs are relatively short (11-35 amino acid)sequences that confer this apparent translocation activity to proteinsand other macromolecular cargo to which they are conjugated, complexedor fused (Derossi et al., 1994; Fawell et al., 1994; Elliott and O'Hare,1997; Schwarze et al., 2000; Snyder and Dowdy, 2001; Bennett et al.,2002).

The highly cationic 11 amino acid residue (YGRKKRRQRRR) PTD from thehuman immunodeficiency virus (HIV-1) TAT protein (Frankel and Pabo,1988; Green and Loewenstein, 1988) has been one of the most well-studiedtranslocating peptides. In-frame fusion proteins containing the TATsequence were shown to direct cellular uptake of proteins that retainedtheir activity intracellularly (Nagahara et al., 1998; Kwon et al.,2000; Becker-Hapak et al., 2001; Jo et al., 2001; Xia et al., 2001; Caoet al., 2002; Joshi et al., 2002; Kabouridis et al., 2002; Peitz et al.,2002). Subsequently, a diverse collection of over 60 full-lengthproteins with functional domains from 15 to 120 kDa have been engineeredto date. Various studies employing TAT-fusion methodologies havedemonstrated transduction in a variety of both primary and transformedmammalian and human cell types, including peripheral blood lymphocytes,diploid fibroblasts, keratinocytes, bone marrow stem cells, osteoclasts,HeLa cells and Jurkat T-cells (Fawell et al., 1994; Nagahara et al.,1998; Gius et al., 1999; Vocero-Akbani et al., 1999, 2000, 2001;Becker-Hapak et al., 2001). Furthermore, in vivo intracellular deliveryby injection of a TAT-b-gal fusion has been demonstrated (Schwarze etal., 1999; Barka et al., 2000). However, intracellular delivery by TATand other peptide domains is inefficient, irreproducible and in manycases results have been misleading due to artifacts caused by fixationprocedures (Richard et al., J. Biol. Chem. 278:585, 2003).

In addition to drug delivery, there are many potential in vitroapplications in areas such as drug discovery and laboratory assays thatcould benefit from improved intracellular delivery of biomolecules andmacromolecular cargo. However, certain challenges remain. For example,even if the biomolecules and macromolecular cargo can be targeted to thedesired cells and endocytosed by the cells, often are not effectivelyreleased from endosomes into the cytosol, but are degraded by lysosomes.These and other challenges are addressed by embodiments of the presentinvention.

SUMMARY OF THE INVENTION

Certain embodiments of the invention include a composition fordelivering an agent to a cell, comprising a bispecific affinity reagentand a pH-responsive, membrane destabilizing polymer. In someembodiments, the bispecific affinity reagent comprises a single protein.In some embodiments, the bispecific affinity reagent comprises anantibody, antibody fragment, or antibody-like molecule. In someembodiments, the bispecific affinity reagent comprises a first affinityreagent covalently linked to a second affinity reagent, wherein thefirst affinity reagent binds to a molecule on the surface of a cell, andthe second affinity reagent binds to an intracellular target. In someembodiments, the first affinity reagent and the second affinity reagentare included in a single polypeptide chain. In some embodiments, thesecond affinity reagent is proteinaceous. In some embodiments, thepolymer is a block copolymer, such as a diblock copolymer, a triblockcopolymer or a higher-order block copolymer.

Certain embodiments of the invention include a composition fordelivering a biomolecular agent such as a therapeutic agent ordiagnostic agent to a cell, comprising a pH-responsive, membranedestabilizing polymer having a plurality of pendant linking groups, anda bispecific affinity reagent. In some embodiments, the bispecificaffinity reagent comprises a first affinity reagent linked to thepolymer and a second affinity reagent linked to the polymer, wherein thefirst affinity reagent binds to a molecule on the surface of a cell, andthe second affinity reagent binds to an intracellular target. In someembodiments, the bispecific affinity reagent comprises a plurality offirst affinity reagents, wherein the plurality of first affinityreagents is linked to the polymer via the pendant linking groups. Insome embodiments, the bispecific affinity reagent comprises a pluralityof second affinity reagents, wherein the plurality of second affinityreagents is linked to the polymer via the pendant linking groups.

Certain embodiments of the invention include a method of altering theactivity of an intracellular target in a cell, comprising contacting acell including an intracellular target having a detectable activity witha composition comprising a pH-responsive, membrane destabilizingcopolymer, and a first affinity reagent covalently linked to a secondaffinity reagent. In some embodiments, the first affinity reagent bindsto a molecule on the surface of the cell thereby bringing the secondaffinity reagent into proximity with the cell surface, wherebyendocytosis of at least the second affinity reagent is facilitated. Insome embodiments, the pH-responsive, membrane destabilizing polymerbecomes membrane-active at acidic pH, thereby causing release of atleast the second affinity reagent from the endosomal compartment of thecell. In some embodiments, the second affinity reagent binds to theintracellular target after release into the interior of the cell,whereby binding of the second affinity reagent to the intracellulartarget detectably agonizes or antagonizes an activity of theintracellular target.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic and chemical structure for a pH-responsivemembrane destabilizing polymer according to an embodiment of the presentinvention.

FIG. 2 illustrates the synthesis of a chain transfer agent according toan embodiment of the present invention.

FIG. 3 illustrates the linking of a bispecific affinity reagent to apH-responsive membrane destabilizing polymer according to an embodimentof the present invention.

FIGS. 4A and 4B depict the results of the use of electrophoresis gels toanalyze the linking of a bispecific affinity reagent to a pH-responsivemembrane destabilizing polymer according to an embodiment of the presentinvention.

FIG. 5 is a chart depicting the results of an assay measuring thepH-dependent membrane disruption capacity of a polymer made according toan embodiment of the present invention.

FIGS. 6A and 6B depict the results of fluorescent microscopic analysisof peptide delivery to cells via a prior art composition and acomposition according to an embodiment of the present invention.

FIGS. 7A and 7B depict the results of a cytotoxity study of acomposition according to an embodiment of the present invention.

FIGS. 8A and 8B depict the results of a bioactivity study of acomposition according to an embodiment of the present invention.

DESCRIPTION OF THE INVENTION

The description, figures, and examples herein relate to intracellulardelivery of bispecific affinity reagents using polymeric deliveryvehicles

Short summaries of certain terms are presented in the description of theinvention. Each term is further explained throughout the description,figures, and examples. Any interpretation of the terms in thisdescription should take into account the full description, figures, andexamples presented herein. All publications recited herein are herebyincorporated by reference.

An affinity reagent is a molecule that binds to an antigen or receptoror other molecule. In some embodiments, an affinity reagent is amolecule that specifically binds to an antigen or receptor or othermolecule. In certain embodiments, some or all of an affinity reagent iscomposed of amino acids (including natural, non-natural, and modifiedamino acids), nucleic acids, or saccharides. In certain embodiments, anaffinity reagent is a small molecule.

Affinity reagents in certain embodiments of the present inventionspecifically bind to molecules or targets. A first affinity reagentbinds to a cell surface antigen, a cell surface receptor, or other cellsurface molecule. A second affinity reagent binds to and influences anintracellular target. A second affinity reagent is a proteinaceousaffinity reagent, an aptamer, or a small molecule therapeutic of greaterthan 500 molecular weight. A bispecific affinity reagent comprises afirst affinity reagent covalently linked to a second affinity reagent. Aproteinaceous affinity reagent is an affinity reagent composed of aminoacids (including natural, non-natural, and modified amino acids).

In some embodiments of the present invention, the first and secondaffinity reagents are proteinaceous and may be present in a singlepeptide or polypeptide chain. In some embodiments, the polypeptide chainis a bispecific antibody.

Bispecific antibodies are well-established in the art as a standardtechnique to create a single polypeptide that binds to two differentdeterminants (Kufer et al., 2004). Bispecific antibodies may be made inmany different formats, including but not limited to quadroma, F(ab′)2,tetravalent, heterodimeric scFv, bispecific scFv, tandem scFv, diabodyand minibody formats, or scFvs appended to or recombinantly fused withwhole antibodies. (Kufer et al, 2004; Holliger and Hudson 2005; Morrisonand Coloma, PCTUS94/11411).

Antibodies for use in the present invention may be raised through anyconventional method, such as through injection of immunogen into miceand subsequent fusions of lymphocytes to create hybridomas. Suchhybridomas may then be used either (a) to produce antibody directly,which is purified and used for chemical conjugation to create abispecific antibody, or (b) to clone cDNAs encoding antibody fragmentsfor subsequent genetic manipulation. To illustrate one method employingthe latter strategy, mRNA is isolated from the hybridoma cells,reverse-transcribed into cDNA using antisense oligo-dT or immunoglobulingene-specific primers, and cloned into a plasmid vector. Clones aresequenced and characterized. They may then be engineered according tostandard protocols to combine the heavy and light chains of eachantibody, separated by a short peptide linker, into a bacterial ormammalian expression vector as previously described to produce arecombinant bispecific antibody, which are then expressed and purifiedaccording to well-established protocols in bacteria or mammalian cells(Kufer et al, 2004; Antibody Engineering: A Practical Approach,McCafferty, Hoogenboom and Chiswell Eds, IRL Press 1996). Antibodies, orother proteinaceous affinity molecules such as peptides, may also becreated through display technologies that allow selection of interactingaffinity reagents through the screening of very large libraries of, forexample, immunoglobulin domains or peptides expressed by bacteriophage(Antibody Engineering: A Practical Approach, McCafferty, Hoogenboom andChiswell Eds, IRL Press 1996). Antibodies of the present invention mayalso be humanized through grafting of human immunoglobulin domains, ormade from transgenic mice or bacteriophage libraries that have humanimmunoglobulin genes/cDNAs.

In some embodiments of the invention, first and second affinity reagentsmay comprise proteinaceous structures other than antibodies that areable to bind to protein targets specifically, including but not limitedto avimers (Silverman et al, 2005), ankyrin repeats (Zahnd et al., 2007)and adnectins (as described in U.S. Pat. No. 7,115,396), and other suchproteins with domains that can be evolved to generate specific affinityfor antigens, collectively referred to as “antibody-like molecules”.Modifications of proteinaceous affinity reagents through theincorporation of unnatural amino acids during synthesis may be used toimprove their properties (see Datta et al., 2002; and Liu et al., 2007).Such modifications may have several benefits, including the addition ofchemical groups that facilitate subsequent conjugation reactions.

In some embodiments, the first or second affinity reagent may be apeptide. In some embodiments, the peptide chain is a bispecific peptide.Peptides can readily be made and screened to create affinity reagentsthat recognize and bind to macromolecules such as proteins (see, forexample, “Phage display of combinatorial peptide and protein librariesand their applications in biology and chemistry”. Current Topics inMicrobiology and Immunology, vol. 243 1999, p. 87-105).

In some embodiments of the invention, proteinaceous first andproteinaceous second affinity reagents are present on two separatepeptide or polypeptide chains. Bispecific affinity reagents may beconstructed by separate synthesis and expression of the first and secondaffinity reagents. A polypeptide bispecific reagent can be expressed astwo separately encoded chains that are linked by disulfide bonds duringproduction in the same host cell, such as, for example, a bispecificscFv or diabody (Kufer et al., 2004; Holliger and Hudson, 2005).Similarly, standard and widely used solid-phase peptide synthesistechnology (see, for example, Handbook of Reagents for OrganicSynthesis, Reagents for Glycoside, Nucleotide, and Peptide Synthesis,David Crich (Ed.) 2005) can be used to synthesize peptides, and chimericbispecific peptides are well known in the art (see, for example, Dickeyet al., J Biol Chem 283:35003, 2008). A bispecific peptide strategy maybe used to combine the first and second first and second affinityreagents in a single peptide chain. Alternatively, polypeptide chains orpeptide chains can be expressed/synthesized separately, purified andthen conjugated chemically to produce the bispecific affinity reagentsof the present invention. Many different formats of antibodies may beused. Whole antibodies, F(ab′)2, F(ab′), scFv, as well as smaller Faband single-domain antibody fragments (Holliger and Hudson, 2005) may allbe used to create the first and second affinity reagents. Followingtheir expression and purification, the first and second affinityreagents can be chemically conjugated to create the bispecific affinityreagent. Many conjugation chemistries may be used to effect thisconjugation, including homofunctional or heterofunctional linkers thatyield ester, amide, thioether, carbon-carbon, or disulfide linkages (seeBioconjugation, Aslam and Dent, Eds, Macmillan, 1998 and chapterstherein). The first and second affinity reagents may also be linked toeach other via the polymer itself (see below).

In some embodiments, first affinity reagents are peptide aptamers. Apeptide aptamer is a peptide molecule that specifically binds to atarget protein, and interferes with the functional ability of thattarget protein (Kolonin et al., Proc. Natl. Acad. Sci. USA 95:14266(1998). Peptide aptamers consist of a variable peptide loop attached atboth ends of a protein scaffold. Such peptide aptamers can often have abinding affinity comparable to that of an antibody (nanomolar range).Due to the highly selective nature of peptide aptamers, they can be usednot only to target a specific protein, but also to target specificfunctions of a given protein (e.g., a signaling function).

Peptide aptamers are usually prepared by selecting the aptamer for itsbinding affinity with the specific target from a random pool or libraryof peptides. Peptide aptamers can be isolated from random peptidelibraries by yeast two-hybrid screens (Xu et al., Proc. Natl. Acad. Sci.USA 94:12473 (1997). They can also be isolated from phage libraries(Hoogenboom et al., Immunotechnology 4:1 (1998) or chemically generatedpeptides/libraries.

First affinity reagents may also be nucleic acid aptamers. Nucleic acidaptamers are nucleic acid oligomers that bind other macromoleculesspecifically; such aptamers that bind specifically to othermacromolecules can be readily isolated from libraries of such oligomersby technologies such as SELEX (see, for example, “SELEX—a(r)evolutionary method to generate high-affinity nucleic acid ligands”,Stoltenburg et al., Biomol. Eng. 24:381, 2007).

In some embodiments, first affinity reagents are oligosaccharides.Certain oligosaccharides are known ligands for certain extracellular orcell surface receptors. For example, Collins et al. describe a syntheticsialoside with affinity for cellular protein CD22. (“High-AffinityLigand Probes of CD22 Overcome the Threshold Set by cis Ligands to Allowfor Binding, Endocytosis, and Killing of B Cells” Collins et al., J.Immunol. 177:2994-3003, 2006).

The first affinity reagent recognizes a cell surface antigen on thetarget cell. The first affinity reagent may be an antibody,antibody-like molecule, or a peptide, such as an integrin-binding RGDpeptide, or a small molecule, such as vitamins, e.g., folate, sugarssuch as lactose and galactose, or other small molecules. The cellsurface antigen may be any cell surface molecule that undergoesinternalization, such as a protein, sugar, lipid head group or otherantigen on the cell surface. Examples of cell surface antigens useful inthe context of the present invention include but are not limited to thetransferrin receptor type 1 and 2, the EGF receptor, HER2/Neu, VEGFreceptors, integrins, CD33, CD19, CD20, CD22 and the asialoglycoproteinreceptor.

First affinity reagents may also include peptide transduction domainssuch as the VP22 uptake peptide from HSV, the HIV TAT protein/peptide,and the antennapedia peptide, the transportan peptide, and polyarginine.Peptide transduction domains are known in the art, as described, forexample, in Snyder EL, Dowdy SF. Recent advances in the use of proteintransduction domains for the delivery of peptides, proteins and nucleicacids in vivo. Expert Opin. Drug Deliv. 2005 January; 2(1):43-51.

In some embodiments, second affinity reagents are the antibodies,antibody-like molecules, peptides, aptamers, or small molecules. In someembodiments, second affinity reagents are dominant negative proteins.Certain dominant negative proteins have been shown to inhibit VEGFpromoter activity. (J Biol Chem 274(44):31565-31570, Oct. 29, 1999)

The second affinity reagent recognizes an intracellular target. This“effector” affinity reagent binds specifically to an intracellularantigen, such as a protein. In some embodiments, the second affinityreagent is proteinaceous, and in certain embodiments is an antibody orantibody-like molecule. Other second affinity reagents include peptides,such as for example the bcl-2 antagonist BH3 peptide (Walsh et al.,2002), and organic macromolecules which by virtue of their size (amolecular weight of >500 g), charge, or other physicochemicalproperties, are unable or poorly able to enter cells. In someembodiments, the second affinity reagent is a nucleic acid aptamer. Thesecond affinity reagent may bind to cytosolic proteins; proteins boundto the inner face of the plasma membrane, or the nuclear, mitochondrialor other membranes in the cell; or nuclear proteins or proteins in othersubcellular compartments. It will be evident to those skilled in the artthat affinity reagents which block critical functions of intracellularsignaling will be good candidates for use as second affinity reagents.Second affinity reagents may directly inhibit the activity of a protein,or block an interaction with a protein's substrate, or they may blockprotein-protein interactions. Also, it is well established that someaffinity reagents are able to rescue defects in intracellular proteins.Such second affinity reagents may therefore act as agonists of theactivity of an intracellular protein. This has been clearly shown in thecase of antibodies against the p53 protein, in which antibodies canincrease the DNA binding and transcriptional activity of the p53 mutantsfound in many cancer cells, which would enable correction of thismutation in the p53 tumor suppressor in cancer cells (see Abarzua etal., Cancer Res. 55(16):3490-4, Aug. 15, 1995). Specific proteinfamilies that may be targeted by the second affinity reagent includekinases, such as the receptor tyrosine kinases including theintracellular domains of the EGF, HER2/neu, PDGF, and VEGF receptors,ion channel receptors, G protein receptors and the intracellular domainsof other cell surface receptors; cellular kinases such as erk, mek, mapkinase (mapK), mapKK, mapKKK, and their substrates such asmitogen-activated protein kinase activated protein kinases (MAPKAPKs),including MAPKAPK2; GTPases such as the h-, k-, and n-ras proteins;phosphatases; proteins involved in apoptosis pathways such as the bcl-2proteins and associated family members; transcription factors; and allother intracellular proteins. In certain embodiments, the proteinaceoussecond affinity reagent is a naturally occurring cellular protein. Insome embodiments, second affinity reagents that are proteins may includethose used to correct a genetic deficiency by exogenously providing themissing protein in association with the polymer vehicles of the presentinvention (such as, for example, glucocerebrosidase deficiency tocorrect symptoms of Gauchers Disease); proteins used to supplement/addprotein function to a cell; dominant-negative proteins; enzymes; andother proteins of therapeutic value.

The first and second affinity reagents may be directly covalently linkedto each other through a covalent bond. Alternatively, the first andsecond affinity reagents are linked to each other through a linker. Thelinker may be a peptide linker or a chemical linker. Specificembodiments contemplate a linker that is a glycine succinate linker, anamino acid linker or combination thereof. Other linkers include, but arenot limited to, a disulfide linker, carbonate linker; imine linkerresulting from reaction of an amine and an aldehyde; phosphate esterlinkers formed by reacting an alcohol with a phosphate group; hydrazonelinkers which are reaction product of a hydrazide and an aldehyde;acetal linkers that are the reaction product of an aldehyde and analcohol; orthoester linkers that are the reaction product of a formateand an alcohol; peptide linkages formed by an amine group, including butnot limited to, at an end of a polymer such as PEG, and a carboxyl groupof a peptide. As described herein, linking does not include ionic orother non-covalent bonding, such as via a biotin-streptavidin linkage.

Bispecific proteinaceous affinity reagents, or component proteinaceousaffinity reagents, may be expressed in bacterial, fungal or mammalianexpression systems and purified by standard chromatographic techniques(Antibody Engineering: A Practical Approach, McCafferty, Hoogenboom andChiswell Eds, IRL Press 1996).

Following their expression/synthesis and purification, the first andsecond affinity reagents are associated with the pH-responsive, membranedestabilizing polymer through a covalent coupling. If the first andsecond affinity reagents are present as one chemical entity, eitherthrough recombinant fusion, or chemical conjugation or association, thebispecific affinity reagent is covalently associated with the polymer.Alternatively, the first and second affinity reagents may be separatelyexpressed and separately covalently associated with the polymer tocreate the bispecific affinity reagent.

A pH-responsive, membrane destabilizing polymer is a polymer that atabout physiologic pH (7.4) undergoes a transition at the lower pHenvironment of the endosome and becomes membrane destabilizing. In somenon-limiting embodiments of the invention the polymers can be made ofhomopolymers of alkyl acrylic acids, such as butyl acrylic acid (BAA) orpropyl acrylic acid (PAA), or can be copolymers of ethyl acrylic acid(EAA) (Jones et al, 2003, Murthy et al., 2003). Polymers of alkyl amineor alkyl alcohol derivatives of maleic-anhydride copolymers with methylvinyl ether or styrene may also be used. In some embodiments, thepolymers can be made as copolymers with other monomers. The addition ofother monomers can enhance the potency of the polymers, or add chemicalgroups with useful functionalities to facilitate association with othermolecular entities, including the bispecific affinity reagent and/orother adjuvant materials such as poly(ethylene glycol). These copolymersmay include, but are not limited to, copolymers with monomers containinggroups that can be crosslinked to the bispecific affinity reagent. In anexemplary embodiment, a copolymer of poly(propylacrylic acid)(PAA)-co-pyridyl disulfide acrylate (PDSA), p(PAA-co-PDSA) (El-Sayed etal., 2005) is conjugated via disulfide exchange between the PDSAcomponent of the polymer and free thiols introduced into theproteinaceous affinity reagent by reaction with Traut's reagent(2-iminothiolane, Pierce Biotechnology, Rockford, Ill.). Similarstrategies using monomers comprising other functionalities to enableconjugation, such as NHS, azide and alkyne monomers, may also be used.

The polymer can also serve as the vehicle through which the first andsecond affinity reagents are linked, in addition to providing itsfunctional properties of pH-responsive membrane destabilization.Conjugation of the first and second affinity reagents to the polymersmay be achieved by using polymers with telechelic ends. These differentends allow separate conjugation of the first and second affinityreagents. In this way, a bispecific affinity reagent is made by linkingthe first and second affinity reagent to either end of the same polymermolecule.

The first and/or second affinity reagent may also be linked to thepolymer via pendant linking groups of the polymer chain. Pendant groupsthat may be used for such conjugation include carboxyl residues such asthose present on the alkyl acrylic acids, as well as chemical groups onother monomers introduced via copolymerization, such as PDSA.

Generally, the various polymers included as constituent moieties of thecompounds of the invention can comprise one or more repeat units—monomer(or monomeric) residues—derived from a process which includespolymerization. Such monomeric residues can optionally also includestructural moieties (or species) derived from post-polymerization (e.g.,derivatization) reactions. Monomeric residues are constituent moietiesof the polymers, and accordingly, can be considered as constitutionalunits of the polymers. Generally, a polymer of the invention cancomprise constitutional units which are derived (directly or indirectlyvia additional processes) from one or more polymerizable monomers.

Generally, each polymer can be a homopolymer (derived frompolymerization of one single type of monomer—having essentially the samechemical composition) or a copolymer (derived from polymerization of twoor more different monomers—having different chemical compositions).Polymers which are copolymers include random copolymer chains or blockcopolymer chains (e.g., diblock copolymer, triblock copolymer,higher-ordered block copolymer, etc). Any given block copolymer chaincan be conventionally configured and effected according to methods knownin the art.

Generally, each polymer can be a linear polymer, or a non-linearpolymer. Non-linear polymers can have various architectures, includingfor example branched polymers, brush polymers, star-polymers, dendrimerpolymers, and can be cross-linked polymers, semi-cross-linked polymers,graft polymers, and combinations thereof.

Polymers of the present invention may be carried out by methodsincluding Atom Transfer Radical Polymerization (ATRP),nitroxide-mediated living free radical polymerization (NMP),ring-opening polymerization (ROP), degenerative transfer (DT), orReversible Addition Fragmentation Transfer (RAFT). In specificembodiments, a polymer can be a prepared by controlled (living) radicalpolymerization, such as reversible addition-fragmentation chain transfer(RAFT) polymerization. Such methods and approaches are generally knownin the art, and are further described herein. Alternatively, a polymercan be a prepared by conventional polymerization approaches, includingconventional radical polymerization approaches.

Generally, a polymer is prepared by a method other than by stepwisecoupling approaches involving a sequence of multiple individualreactions (e.g., such as known in the art for peptide synthesis or foroligonucleotide synthesis). The backbone of the membrane-destabilizingpolymer is not a peptidic polymer, a nucleic acid polymer, or a lipidpolymer. In contrast, for clarity, notwithstanding and without prejudiceto the foregoing, the affinity reagents and/or other biomolecular agentsof the inventions can be an amino acid polymer (e.g., a peptide) or anucleic acid polymer (e.g., an oligonucleotide).

Generally, polymers prepared by controlled (living) radicalpolymerization, such as reversible addition-fragmentation chain transfer(RAFT) polymerization, may include moieties other than the monomericresidues (repeat units). For example, and without limitation, suchpolymers may include polymerization-process-dependent moieties at theα-end or at the ω-end of the polymer chain. Typically, for example, apolymer chain derived from controlled radical polymerization such asRAFT polymerization may further comprise a radical source residuecovalently coupled with the α-end thereof. For example, the radicalsource residue can be an initiator residue, or the radical sourceresidue can be a leaving group of a reversible addition-fragmentationchain transfer (RAFT) agent. Typically, as another example, a polymerderived from controlled radical polymerization such as RAFTpolymerization may further comprise a chain transfer residue covalentlycoupled with the ω-end thereof. For example, a chain transfer residuecan be a thiocarbonylthio moiety having a formula —SC(═S)Z, where Z isan activating group. Typical RAFT chain transfer residues are derivedfrom radical polymerization in the presence of a chain transfer agentselected from xanthates, dithiocarbamates, dithioesters, andtrithiocarbonates. The process-related moieties at α-end or at the ω-endof the polymer or between blocks of different polymers can comprise orcan be derivatized to comprise functional groups, e.g., suitable forcovalent linking, etc.

Further aspects of the polymers are disclosed in the followingparagraphs, including preferred polymerizable monomers from which therepeat units of the polymers are derived.

In preferred embodiments, the polymers can comprise repeat units derivedfrom ethylenically unsaturated monomers. The term “ethylenicallyunsaturated monomer” is defined herein as a compound having at least onecarbon double or triple bond. The non-limiting examples of theethylenically unsaturated monomers are: an alkyl (alkyl)acrylate, aalkyl methacrylate, an alkylacrylic acid, an N-alkylacrylamide, amethacrylamide, a styrene, an allylamine, an allylammonium, adiallylamine, a diallylammonium, an n-vinyl formamide, a vinyl ether, avinyl sulfonate, an acrylic acid, a sulfobetaine, a carboxybetaine, aphosphobetaine, or maleic anhydride.

In various embodiments, any monomer suitable for providing the polymersdescribed herein may be used to effect the invention. In someembodiments, monomers suitable for use in the preparation of polymersprovided herein include, by way of non-limiting example, one or more ofthe following monomers: methyl methacrylate, ethyl acrylate, propylmethacrylate (all isomers), butyl methacrylate (all isomers),2-ethylhexyl methacrylate, isobornyl methacrylate, methacrylic acid,benzyl methacrylate, phenyl methacrylate, methacrylonitrile,alpha-methylstyrene, methyl acrylate, ethyl acrylate, propyl acrylate(all isomers), butyl acrylate (all isomers), 2-ethylhexyl acrylate,isobornyl acrylate, acrylic acid, benzyl acrylate, phenyl acrylate,acrylonitrile, styrene, acrylates and styrenes selected from glycidylmethacrylate, 2-hydroxyethyl methacrylate, hydroxypropyl methacrylate(all isomers), hydroxybutyl methacrylate (all isomers),N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate,triethyleneglycol methacrylate, itaconic anhydride, itaconic acid,glycidyl acrylate, 2-hydroxyethyl acrylate, hydroxypropyl acrylate (allisomers), hydroxybutyl acrylate (all isomers), N,N-dimethylaminoethylacrylate, N,N-diethylaminoethyl acrylate, triethyleneglycol acrylate,methacrylamide, N-methylacrylamide, N,N-di methylacrylamide,N-tert-butylmethacrylamide, N-n-butylmethacrylamide,N-methylolacrylamide, N-ethylolacrylamide, vinyl benzoic acid (allisomers), diethylaminostyrene (all isomers), alpha-methylvinyl benzoicacid (all isomers), diethylamino alpha-methylstyrene (all isomers),p-vinylbenzenesulfonic acid, p-vinylbenzene sulfonic sodium salt,trimethoxysilylpropyl methacrylate, triethoxysilylpropyl methacrylate,tributoxysilylpropyl methacrylate, di methoxymethylsilylpropylmethacrylate, diethoxymethylsilylpropylmethacrylate,dibutoxymethylsilylpropyl methacrylate, diisopropoxymethylsilylpropylmethacrylate, dimethoxysilylpropyl methacrylate, diethoxysilylpropylmethacrylate, dibutoxysilylpropyl methacrylate, diisopropoxysillpropylmethacrylate, trimethoxysilylpropyl acrylate, triethoxysilylpropylacrylate, tributoxysilylpropyl acrylate, dimethoxymethylsilylpropylacrylate, diethoxymethylsilylpropyl acrylate, dibutoxymethylsilylpropylacrylate, diisopropoxymethylsilylpropyl acrylate, dimethoxysilylpropylacrylate, diethoxysilylpropyl acrylate, dibutoxysilylpropyl acrylate,diisopropoxysilylpropyl acrylate, vinyl acetate, vinyl butyrate, vinylbenzoate, vinyl chloride, vinyl fluoride, vinyl bromide, maleicanhydride, N-arylmaleimide, N-phenylmaleimide, N-alkylmaleimide,N-butylimaleimide, N-vinylpyrrolidone, N-vinylcarbazole, butadiene,isoprene, chloroprene, ethylene, propylene, 1,5-hexadienes,1,4-hexadienes, 1,3-butadienes, 1,4-pentadienes, vinylalcohol,vinylamine, N-alkylvinylamine, allylamine, N-alkylallylamine,diallylamine, N-alkyldiallylamine, alkylenimine, acrylic acids,alkylacrylates, acrylamides, methacrylic acids, alkylmethacrylates,methacrylamides, N-alkylacrylamides, N-alkylmethacrylamides, styrene,vinylnaphthalene, vinyl pyridine, ethylvinylbenzene, aminostyrene,vinylimidazole, vinylpyridine, vinylbiphenyl, vinylanisole,vinylimidazolyl, vinylpyridinyl, vinylpolyethyleneglycol,dimethylaminomethylstyrene, trimethylammonium ethyl methacrylate,trimethylammonium ethyl acrylate, dimethylamino propylacrylamide,trimethylammonium ethylacrylate, trimethylanunonium ethyl methacrylate,trimethylammonium propyl acrylamide, dodecyl acrylate, octadecylacrylate, or octadecyl methacrylate monomers, or combinations thereof.

In some embodiments, polymers can be derived from certain specificmonomers and combinations of monomers, for example, for use inconnection with various embodiments, such as for uses associated withproteinaceous compositions. Such preferred polymers are described below.

Generally, polymers can include repeat units derived from functionalizedmonomers, including versions of the aforementioned monomers. Afunctionalized monomer, as used herein, can include a monomer comprisinga masked (protected) or non-masked (unprotected) functional group, e.g.,a group to which other moieties, can be covalently attached followingthe polymerization. The non-limiting examples of such groups are primaryamino groups, carboxyls, thiols, hydroxyls, azides, and cyano groups.Several suitable masking groups are available (see, e.g., T. W. Greene &P. G. M. Wuts, Protective Groups in Organic Synthesis (2nd edition) J.Wiley & Sons, 1991. P. J. Kocienski, Protecting Groups, Georg ThiemeVerlag, 1994).

As used herein, a “block” copolymer refers to a structure comprising oneor more sub-combination of constitutional or monomeric units. In someembodiments, the block copolymer is a diblock copolymer, a tri-blockcopolymer or a higher-ordered block copolymer. For example, a diblockcopolymer can comprise two blocks; a schematic generalization of such apolymer is represented by the following: [Aa/Bb/Cc/ . . . ]m-[Xx/Yy/Zz/. . . ]n, wherein each letter stands for a constitutional or monomericunit, and wherein each subscript to a constitutional unit represents themole fraction of that unit in the particular block, the three dotsindicate that there may be more (there may also be fewer) constitutionalunits in each block and m and n indicate the molecular weight (or weightfraction) of each block in the diblock copolymer. As suggested by suchschematic representation, in some instances, the number and the natureof each constitutional unit is separately controlled for each block. Theschematic is not meant to, and should not be construed to, infer anyrelationship whatsoever between the number of constitutional units orbetween the number of different types of constitutional units in each ofthe blocks. Nor is the schematic meant to describe any particular numberor arrangement of the constitutional units within a particular block. Ineach block the constitutional units may be disposed in a purely random,an alternating random, a regular alternating, a regular block or arandom block configuration unless expressly stated to be otherwise. Apurely random configuration, for example, may have the form:x-x-y-z-x-y-y-z-y-z-z-z . . . An exemplary alternating randomconfiguration may have the form: x-y-x-z-y-x-y-z-y-x-z . . . , and anexemplary regular alternating configuration may have the form:x-y-z-x-y-z-x-y-z . . . An exemplary regular block configuration mayhave the following general configuration: . . . x-x-x-y-y-y-z-z-z-x-x-x. . . , while an exemplary random block configuration may have thegeneral configuration: . . . x-x-x-z-z-x-x-y-y-y-y-z-z-z-x-x-z-z-z- . .. In a gradient polymer, the content of one or more monomeric unitsincreases or decreases in a gradient manner from the α end of thepolymer to the ω end. In none of the preceding generic examples is theparticular juxtaposition of individual constitutional units or blocks orthe number of constitutional units in a block or the number of blocksmeant nor should they be construed as in any manner bearing on orlimiting the actual structure of block copolymers forming the polymericcarrier of this invention.

As used herein, the brackets enclosing the constitutional units are notmeant and are not to be construed to mean that the constitutional unitsthemselves form blocks. That is, the constitutional units within thesquare brackets may combine in any manner with the other constitutionalunits within the block, i.e., purely random, alternating random, regularalternating, regular block or random block configurations. The blockcopolymers described herein are, optionally, alternate, gradient orrandom block copolymers.

A unimer or monoblock polymer is a synthetic product of a singlepolymerization step. The term monoblock polymer includes a copolymersuch as a random copolymer (i.e. a product of polymerization of morethan one type of monomers) and a homopolymer (i.e. a product ofpolymerization of a single type of monomers).

Methods for preparing polymers are described below, and are generallyapplicable for, but not be limiting of, the polymers described herein.

Membrane Destabilizing Polymers

As used herein, “membrane-destabilizing” or “destabilizing of a cellmembrane” refers to the ability of a composition comprising one or moremembrane destabilizing polymers to elicit a permeability change in acellular membrane structure (e.g., an endosomal membrane) so as topermit macromolecules or biomolecules, in combination with orindependent of a polymer or micellic assembly described herein, to entera cell or to exit a cellular vesicle (e.g., an endosome). Thispermeability change can be functionally defined by the polymer'sactivity in assays that, for example, measure red blood cell lysis(hemolysis) or by release of nucleic acid or peptide molecules fromcellular endosomal compartments. Complete membrane disruption refers toa mechanism thought to result in the lysis of the endosome.

In certain embodiments, the polymer can be or comprise at least one ormore polymers (including for example as regions or segments, such as ablock of a block copolymer) which comprise a membrane destabilizingpolymer. In other embodiments, the polymer can be or comprise at leastone membrane disruptive polymer. Preferred polymers provided herein canbe a cellular membrane destabilizing polymer (e.g., is disruptive of acellular membrane), such as, by way of non-limiting example, anextracellular membrane, an intracellular membrane, a vesicle, anorganelle, an endosome, a liposome, or a red blood cell. Preferably, incertain instances, wherein a polymer described herein is in contact witha cellular membrane, it disrupts the membrane and enters theintracellular environment. In specific embodiments, a polymer providedherein is hemolytic. In specific embodiments, a polymer provided hereinis endosomolytic.

The membrane destabilizing or membrane disruptive polymer can be a pHsensitive polymer having membrane destabilizing activity or membranedisrupting activity at a desired pH. In some embodiments, membranedestabilizing polymers (e.g., copolymers) or membrane destabilizingblock copolymers provided herein are membrane destabilizing (e.g., in anaqueous medium) at an endosomal pH. In some embodiments, the membranedestabilizing block copolymers are membrane destabilizing (e.g., in anaqueous medium) at a pH of about 6.5 or lower, preferably at a pHranging from about 5.0 to about 6.5, or at a pH of about 6.2 or lower,preferably at a pH ranging from about 5.0 to about 6.2, or at a pH ofabout 6.0 or lower, preferably at a pH ranging from about 5.0 to about6.0.

Preferably, in each case, the membrane destabilizing polymer can havemembrane destabilizing activity or membrane disrupting activity at adesired quantity (e.g., concentration) of polymer. A membranedestabilizing or membrane disruptive characteristic of a polymer can bedetermined by suitable assays known in the art. For example,membrane-destabilizing activity or membrane-disruptive activity of apolymer can be determined in an in vitro cell assay such as the redblood cell hemolysis assay. An endosomolytic polymer activity can bedetermined in an in vitro cell assay.

In general, the membrane destabilizing polymer is composed of monomericresidues with particular properties. Anionic monomeric residues comprisea species charged or chargeable to an anion, including a protonatableanionic species. Anionic monomeric residues can be anionic at anapproximately neutral pH of 7.2-7.4. Cationic monomeric residuescomprise a species charged or chargeable to a cation, including adeprotonatable cationic species. Cationic monomeric residues can becationic at an approximately neutral pH of 7.2-7.4. Hydrophobicmonomeric residues comprise a hydrophobic species. Hydrophilic monomericresidues comprise a hydrophilic species.

Preferably in this regard, for example, the polymer can be or compriseat least one polymer chain which is hydrophobic. Preferably in thisregard, the polymer can be or comprise at least one polymer chain whichincludes a plurality of (anionic) monomeric residues. In this regard,for example, the polymer can preferably be or comprise at least onepolymer chain which includes (i) a plurality of hydrophobic monomericresidues having a hydrophobic species, and (ii) a plurality of (anionic)monomeric residues which can preferably be anionic at approximatelyneutral pH, and substantially neutral or non-charged at an endosomal pHor weakly acidic pH.

In such aforementioned embodiments, the polymer can further comprise aplurality of cationic species. Accordingly, for example, the polymer canbe or comprise at least one polymer chain which includes a plurality ofanionic monomeric residues (e.g., having species that are anionic atabout neutral pH), and a plurality of hydrophobic monomeric residues(e.g., having hydrophobic species), and optionally a plurality ofcationic monomeric residues (e.g., having species that are cationic atabout neutral pH). In such embodiments, and as discussed further below,the polymer can be or comprise at least one polymer chain which ischarge modulated, and preferably charge balanced—being substantiallyoverall neutral in charge.

In some embodiments the membrane destabilizing or membrane destabilizingpolymer can be block copolymer, and can comprise a membranedestabilizing segment (e.g., as a block or region of the polymer). Themembrane destabilizing segment can comprise a plurality of anionicmonomeric residues (e.g., having species that are anionic at aboutneutral pH), and a plurality of hydrophobic monomeric residues (e.g.,having hydrophobic species), and optionally a plurality of cationicmonomeric residues (e.g., having species that are cationic at aboutneutral pH). In such embodiments, the segment (e.g., block or region)can be hydrophobic considered in the aggregate. In such embodiments, theblock copolymer may further comprise a hydrophilic segment.

As a general, non-limiting example, a composition can comprise polymerswhich comprise a block copolymer, where the block copolymer comprisesone or more polymer chains (e.g., with each such chain defining apolymer block), with at least polymer chain being or comprising amembrane destabilizing polymer (e.g., such as an endosomal membranedestabilizing polymer). For example, in one orientation, the blockcopolymer can preferably comprise a first polymer chain defining a firstblock A of the copolymer, and a second membrane destabilizing polymerchain defining a second block B of the copolymer. For example, the blockcopolymer can comprise a first polymer chain defining a first block A ofthe copolymer, which is hydrophilic, and a second polymer chain defininga second block B of the copolymer which includes (i) a plurality ofhydrophobic monomeric residues, and (ii) a plurality of anionicmonomeric residues being anionic at serum physiological pH, and beingsubstantially neutral or non-charged at an endosomal pH.

In some embodiments of the invention, the polymer can be or comprise atleast one polymer chain which includes a plurality of anionic monomericresidues, a plurality of hydrophobic monomeric residues, and optionallya plurality of cationic monomeric residues in ratios adapted to enhancemembrane destabilizing or membrane destabilizing activity of the polymerchain. For example and without limitation, in such embodiments at pH7.4, the ratio of hydrophobic: (anionic+cationic) species ranges fromabout 1:2 to about 3:1, and the ratio of anionic: cationic speciesranges from about 1:0 to about 1:4. In other such embodiments, at pH7.4, the ratio of hydrophobic: (anionic+cationic) species ranges fromabout 1:1 to about 2:1, and the ratio of anionic: cationic speciesranges from about 4:1 to about 1:4.

Generally, the polymer can be or comprise at least one polymer chainwhich is charge modulated, for example including hydrophobic monomericresidues together with both anionic monomeric residues and cationicmonomeric residues. The relative ratio of anionic monomeric residues andcationic monomeric residues can be controlled to achieve a desiredoverall charge characteristic. In preferred embodiments, for example,such polymer or polymer chain can be charge balanced—having asubstantially neutral overall charge in an aqueous medium atphysiological pH (e.g., pH 7.2 to 7.4).

Embodiments comprising a block copolymer, in which at least one block isor comprises a membrane destabilizing polymer, such as a hydrophobicmembrane destabilizing polymer, can comprise one or more further polymerchains as additional blocks of the block copolymer. Generally, suchfurther polymer blocks are not narrowly critical, and can be or comprisea polymer chain which is hydrophilic, hydrophobic, amphiphilic, and ineach case, which is neutral, anionic or cationic in overall chargecharacteristics.

In embodiments of the invention, the polymer can be or comprise apolymer chain which is adapted to facilitate one or more additionalconstituent components and/or functional features of the compound orcomposition of the invention. For example, such polymer chain cancomprise an end functional group (e.g., on the alpha end or omega end ofthe polymer chain) adapted for covalently linking, directly orindirectly, to an affinity reagent, or a shielding agent. Additionallyor alternatively, such polymer chain can comprise one or more monomericresidues having a pendant functional group adapted for conjugating to anagent. Such conjugatable monomeric residues can be effected forcovalently linking, directly or indirectly, to an affinity reagent, ashielding agent, or other biomolecular agent. Additionally oralternatively, such polymer chain can comprise one or more monomericresidues having a shielding species. For example, shielding monomericresidues can be derived directly from a polymerization reaction whichincludes polymerizable monomers comprising a shielding moiety. Shieldingagents include poly ethylene glycol monomers and/or polymers.Additionally or alternatively, such polymer chain can comprise one ormore monomeric residues having a two or more pendant functionalgroups—suitable for crosslinking between polymer chains. Suchcrosslinking monomeric residues can be a constituent moiety of acrosslinked polymer or polymer chain, as derived directly from apolymerization reaction which includes one or more polymerizablemonomers comprising a multi-functional (e.g., bis-functional)crosslinking monomer. Various additional aspects and specific featuresof such agents and such moieties, including biomolecular agents,targeting agents, shielding agents conjugating moieties and crosslinkingmoieties are discussed herein.

Generally, one or more blocks of a block copolymer can be a randomcopolymer block which comprises two or more compositionally distinctmonomeric residues.

Generally, a single monomeric residue can include multiple moietieshaving different functionality—e.g., can comprise hydrophobic species aswell as anionic species, or e.g., can comprise hydrophobic species aswell as cationic species, or e.g., can comprise anionic species as wellas cationic species. Hence, in any embodiment, the polymer can be or cancomprise a polymer comprising a monomeric residue such as an anionichydrophobic monomeric residue—which includes hydrophobic species andanionic species (e.g., species which are anionic at about neutral pH).

Anionic monomeric residues can preferably comprise a protonatableanionic species. Considered in the aggregate, as incorporated into apolymer chain, such anionic monomeric residues can be substantiallyanionic at a pH of or greater than 7.0 and substantially neutral(non-charged) at pH of or less than 6.0. Preferably, such anionicmonomeric residues can have a pKa ranging from about 5.5 to about 6.8.Anionic monomeric residues can independently comprise a plurality ofmonomeric residues having a protonatable anionic species selected fromcarboxylic acid, sulfonamide, boronic acid, sulfonic acid, sulfinicacid, sulfuric acid, phosphoric acid, phosphinic acid, and phosphorousacid groups, and combinations thereof. Preferred anionic monomericresidues can be derived from polymerization of a (C₂-C₈) alkylacrylicacid.

Hydrophobic monomeric residues can be charged or noncharged, generally.Some embodiments include neutral (non-charged) hydrophobic monomericresidues. In some embodiments, polymer chains can independently comprisea plurality of monomeric residues having a hydrophobic species selectedfrom (C₂-C₈) alkyl, (C₂-C₈) alkenyl, (C₂-C₈) alkynyl, aryl, andheteroaryl (each of which may be optionally substituted). In certainembodiments, the plurality of monomeric residues can be derived frompolymerization of (C₂-C₈) alkyl-ethacrylate, a (C₂-C₈)alkyl-methacrylate, or a (C₂-C₈) alkyl-acrylate (each of which may beoptionally substituted).

Cationic monomeric residues can preferably comprise a deprotonatablecationic species. Considered in the aggregate, as incorporated into apolymer chain, such cationic monomeric residues can be substantiallycationic at a pH of or greater than 7.0. Preferably, such cationicmonomeric residues can have a pKa ranging from about 6.5 to about 9.0.Cationic monomeric residues can independently comprise a plurality ofmonomeric residues having a deprotonatable cationic species selectedfrom the group consisting of acyclic amine, acyclic imine, cyclic amine,cyclic imine, and nitrogen-containing heteroaryl. Preferred cationicmonomeric residues can be derived from polymerization of, in each caseoptionally substituted,(N,N-di(C1-C6)alkyl-amino(C1-C6)alkyl-ethacrylate,N,N-di(C1-C6)alkyl-amino(C1-C6)alkyl-methacrylate, orN,N-di(C1-C6)alkyl-amino(C1-C6)alkyl-acrylate.

Particularly preferred polymers or polymer chains can be block copolymerwhich can comprise or consist essentially of two or more blocksrepresented by formula I,

-   -   where        -   A0, A1, A2, A3 and A4 are each selected from the group            consisting of —C—C—, —C—, —C(O)(C)aC(O)O—, —O(C)aC(O)— and            —O(C)bO—,        -   a is an integer ranging from 1-4; and        -   b is an integer ranging from 2-4;        -   Y4 is selected from the group consisting of hydrogen,            (1C-10C)alkyl, (3C-6C)cycloalkyl, O-(1C-10C)alkyl,            —C(O)O(1C-10C)alkyl, C(O)NR6(1C-10C) and aryl, any of which            is optionally substituted with one or more fluorine groups;        -   Y0, Y1 and Y2 are each independently selected from the group            consisting of a covalent bond, (1C-10C)alkyl-,            —C(O)O(2C-10C) alkyl-, —OC(O)(1C-10C)alkyl-,            —O(2C-10C)alkyl- and            —S(2C-10C)alkyl-1-C(O)NR6(2C-10C)alkyl-;        -   Y3 is selected from the group consisting of a covalent bond,            (1C-10C)alkyl and (6C-10C)aryl; wherein tetravalent carbon            atoms of A1-A4 that are not fully substituted with R1-R5 and            Y0-Y4 are completed with an appropriate number of hydrogen            atoms;        -   each R1, R2, R3, R4, R5, and R6 are independently selected            from the group consisting of hydrogen, —CN, alkyl, alkynyl,            heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and            heteroaryl, any of which may be optionally substituted with            one or more fluorine atoms;        -   Q0 is a residue selected from the group consisting of            residues which are hydrophilic at physiologic pH and are at            least partially positively charged at physiologic pH (e.g.,            amino, alkylamino, ammonium, alkylammonium, guanidine,            imidazolyl, pyridyl, or the like); at least partially            negatively charged at physiologic pH but undergo protonation            at lower pH (e.g., carboxyl, sulfonamide, boronate,            phosphonate, phosphate, or the like); substantially neutral            (or non-charged) at physiologic pH (e.g., hydroxy,            polyoxylated alkyl, polyethylene glycol, polypropylene            glycol, thiol, or the like); at least partially zwitterionic            at physiologic pH (e.g., a monomeric residue comprising a            phosphate group and an ammonium group at physiologic pH);            conjugatable or functionalizable residues (e.g. residues            that comprise a reactive group, e.g., azide, alkyne,            succinimide ester, tetrafluorophenyl ester,            pentafluorophenyl ester, p-nitrophenyl ester, pyridyl            disulfide, or the like); or hydrogen;        -   Q1 is a residue which is hydrophilic at physiologic pH, and            is at least partially positively charged at physiologic pH            (e.g., amino, alkylamino, ammonium, alkylammonium,            guanidine, imidazolyl, pyridyl, or the like); at least            partially negatively charged at physiologic pH but undergoes            protonation at lower pH (e.g., carboxyl, sulfonamide,            boronate, phosphonate, phosphate, or the like);            substantially neutral at physiologic pH (e.g., hydroxy,            polyoxylated alkyl, polyethylene glycol, polypropylene            glycol, thiol, or the like); or at least partially            zwitterionic at physiologic pH (e.g., a monomeric residue            comprising a phosphate group and an ammonium group at            physiologic pH);        -   Q2 is a residue which is positively charged at physiologic            pH, including but not limited to amino, alkylamino,            ammonium, alkylammonium, guanidine, imidazolyl, and pyridyl;        -   Q3 is a residue which is negatively charged at physiologic            pH, but undergoes protonation at lower pH, including but not            limited to carboxyl, sulfonamide, boronate, phosphonate, and            phosphate;        -   m is a number ranging from equal to 0 to less than 1.0            (e.g., 0 to about 0.49);        -   n is a number ranging from greater than 0 to 1.0 (e.g.,            about 0.51 to about 1.0);        -   the sum of (m+n)=1        -   p is a number ranging from about 0.1 to about 0.9 (e.g.,            about 0.2 to about 0.5);        -   q is a number ranging from about 0.1 to about 0.9 (e.g.,            about 0.2 to about 0.5);        -   r is a number ranging from 0 to about 0.8 (e.g., 0 to about            0.6);        -   the sum of (p+q+r)=1;        -   v ranges about 1 to about 25 kDa; and,        -   w ranges from about 1 to about 50 kDa.

In some embodiments, the number or ratio of monomeric residuesrepresented by p and q are within about 30% of each other, about 20% ofeach other, about 10% of each other, or the like. In specificembodiments, p is substantially the same as q. In certain embodiments,at least partially charged generally includes more than a trace amountof charged species, including, e.g., at least 20% of the residues arecharged, at least 30% of the residues are charged, at least 40% of theresidues are charged, at least 50% of the residues are charged, at least60% of the residues are charged, at least 70% of the residues arecharged, or the like.

In certain embodiments, m is 0 and Q1 is a residue which is hydrophilicand substantially neutral (or non-charged) at physiologic pH. In someembodiments, substantially non-charged includes, e.g., less than 5% arecharged, less than 3% are charged, less than 1% are charged, or thelike. In certain embodiments, m is 0 and Q1 is a residue which ishydrophilic and at least partially cationic at physiologic pH. Incertain embodiments, m is 0 and Q1 is a residue which is hydrophilic andat least partially anionic at physiologic pH. In certain embodiments, mis >0 and n is >0 and one of and Q0 or Q1 is a residue which ishydrophilic and at least partially cationic at physiologic pH and theother of Q0 or Q1 is a residue which is hydrophilic and is substantiallyneutral at physiologic pH. In certain embodiments, m is >0 and n is >0and one of and Q0 or Q1 is a residue which is hydrophilic and at leastpartially anionic at physiologic pH and the other of Q0 or Q1 is aresidue which is hydrophilic and is substantially neutral at physiologicpH. In certain embodiments, m is >0 and n is >0 and Q1 is a residuewhich is hydrophilic and at least partially cationic at physiologic pHand Q0 is a residue which is conjugatable or functionalizable residues.In certain embodiments, m is >0 and n is >0 and Q1 is a residue which ishydrophilic and substantially neutral at physiologic pH and Q0 is aresidue which is h conjugatable or functionalizable residues.

Exemplary but non-limiting polymers of this invention can be or comprisea polymer chain which is a random copolymer represented as compound 1,optionally with one or more counterions.

The constitutional units of compound 1 can be derived from thepolymerizable monomers N,N-dimethylaminoethylmethacrylate (DMAEMA or“D”), propylacrylic acid (PAA or “P”) and butyl methacrylate (BMA or“B”), represented respectively as follows:

For the polymer chain represented by compound 1, p, q and r representthe mole fraction of each constitutional unit within the polymer chain,and can have the values described below.

The polymer can be a chain of compound 1, or can comprise a chain ofcompound 1 as one block of a block copolymer. For example, in oneembodiment, the polymer can be a block copolymer comprising compound 1as a membrane disrupting polymer block and one or more additionalblocks. A diblock copolymer for example, can be represented by [A]v-[1]wwhere [A] represents a second block (e.g., a hydrophilic block or anamphiphilic block), and the letters v and w represent the molecularweight (number average) of the respective blocks in the copolymer.

For example, a polymer can comprise a block copolymer having two or moreblocks, including blocks having a structure represented as follows (withappropriate counterions):

The constitutional units of compound 2 can be derived from thepolymerizable monomers O—(C1-C6 alkyl)polyethyleneglycol-methacrylate(PEGMA) (first block as shown) and from the polymerizable monomersDMAEMA, PAA, and BMA as described above in connection with compound 1(second block as shown). Letters p, q and r represent the mole fractionof each constitutional unit within the second block (as shown) and canhave the values described below. The letters v and w represent themolecular weight (number average) of each block in the block copolymerand can have the values described below.

Particularly preferred polymers of the invention can comprise a blockcopolymer having two or more blocks, including blocks having a structurerepresented as:[DMAEMA]_(v)-[B_(p)−/−P_(q)−/−D_(r)]_(w)  3[PEGMA]_(v)-[B_(p)−/−P_(q)−/−D_(r)]_(w)  4[PEGMA_(m)−/−DMAEMA_(n)]_(v)-[B_(p)−/−P_(q)−/−D_(r)]_(w)  5[PEGMA_(m)−/−MAA(NHS)_(n)]_(v)-[B_(p)−/−P_(q)−/−D_(r)]_(w)  6[DMAEMA_(m)−/−MAA(NHS)_(n)]_(v)-[B_(p)−/−P_(q)−/−D_(r)]_(w)  7[HPMA_(m)−/−PDSM_(n)]_(v)-[B_(p)−/−P_(q)−/−D_(r)]_(w)  8[PEGMA_(m)−/−PDSM_(n)]_(v)-[B_(p)−/−P_(q)−/−D_(r)]_(w)  9where B is butyl methacrylate residue; P is propylacrylic acid residue;D, DMAEMA are each dimethylaminoethyl methacrylate residue; PEGMA ispolyethyleneglycol methacrylate residue (e.g., with 1-20 ethylene oxideunits, such as illustrated in compound 2, or 4-5 ethylene oxide units,or 7-8 ethylene oxide units); MAA(NHS) is methylacrylic acid-N-hydroxysuccinimide residue; HPMA is N-(2-hydroxypropyl) methacrylamide residue;and PDSM is pyridyl disulfide methacrylate residue.

Generally, for each of the polymers comprising compounds 1 through 9,each of m, n, p, q, r, w and v are numbers. Preferably:

-   -   p is a number ranging from about 0.1 to about 0.9 (e.g., about        0.2 to about 0.5);    -   q is a number ranging from about 0.1 to about 0.9 (e.g., about        0.2 to about 0.5);    -   r is a number ranging from 0 to about 0.8 (e.g., 0 to about        0.6);    -   the sum of (p+q+r)=1;    -   v ranges about 5 to about 25 kDa; and,    -   w ranges from about 5 to about 50 kDa.        In some specific embodiments, the ratio of w:v ranges from about        1:1 to about 5:1.

Polymers 1-9 are representative examples of polymers suitable for use inconnection with the present invention. In certain priority applications,the polymer PRx0729v6 is used interchangeably with the polymer P7v6.Other polymers can also be used, including structurally related polymers(such as variations in molecular weights and/or monomeric residueratios). In some embodiments, the constitutional unit(s) of the firstblock (as shown) are controlled to effect a first block (as shown) whichis or comprises a constitutional unit that is neutral (e.g., PEGMA),cationic (e.g., DMAEMA), anionic (e.g., PEGMA-NHS, where the NHS ishydrolyzed to the acid, or acrylic acid), ampholytic (e.g., DMAEMA-NHS,where the NHS is hydrolyzed to the acid), or zwiterrionic (for example,poly[2-methacryloyloxy-2′ trimethylammoniumethyl phosphate]). In someembodiments, polymers comprising pyridyl disulfide functionality in thefirst block (as shown), e.g., [PEGMA-PDSM]-[B-P-D], that can be and isoptionally reacted with a thiolated biomolecular agent [such as athiolated siRNA to form a polymer-siRNA conjugate].

Shielding/Solubilizing Agents

Generally, the various polymers (or polymer chains included asconstituent moieties such as blocks of a block copolymer) of thecompounds of the invention can comprise one or more shielding agentand/or solubilizing agent. The shielding agent can be effective forimproving solubility of the polymer chain and can be effective forsteric shielding of a therapeutic agent (e.g., polynucleotide, peptide,etc.). The shielding agent can also be effective for enhancing thestability of the therapeutic agent (e.g., polynucleotide or peptide,etc.) against enzymatic digestion in plasma. The shielding agent canalso be effective for reducing toxicity of the certain compositions(e.g., compositions comprising polynucleotides). In some embodiments,the shielding agent can be a polymer comprising a plurality of neutralhydrophilic monomeric residues. The shielding polymer can be covalentlycoupled to a membrane destabilizing polymer, directly or indirectly,through an end group of the polymer or through a pendant functionalgroup of one or more monomeric residues of the polymer. In someembodiments, a plurality of monomeric residues of the polymer chain canhave a shielding species; preferably, such shielding species is apendant moiety from a polymerizable monomer (from which the shieldingmonomeric residues are derived). For example, the polymer can comprise aplurality of monomeric residues having a pendant group comprising ashielding oligomer.

A preferred shielding/solubilizing polymer can be a polyethylene glycol(PEG) oligomer (e.g., having 20 or less repeat units) or polymer (e.g.,having more than 20 repeat units). In certain embodiments, one block ofa block copolymer can be or comprises a polyethylene glycol (PEG)oligomer or polymer—for example, covalently coupled to the alpha end orthe omega end of the membrane destabilizing block of the copolymer. Inanother embodiment, a polyethylene glycol (PEG) oligomer or polymer canbe covalently coupled to the polymer through a conjugating monomericresidue having a species which includes a functional group suitable forlinking, directly or indirectly, to the polyethylene glycol oligomer orpolymer. In another embodiment, the monomeric residue can be derivedfrom a polymerizable monomer which includes a polyethylene glycololigomer pendant to the monomer (e.g., PEGMA as described above).

In one general approach, PEG chains or blocks are covalently coupled toa membrane destabilizing polymer chain. For such embodiments, forexample, PEG chains or blocks can have molecular weights rangingapproximately from 1,000 to approximately 30,000. In some embodiments,the PEG is effective as (i.e., is incorporated into) a second block of ablock copolymer. For example, PEG can be a second block coupledcovalently to a block comprising a membrane destabilizing polymer. Insome embodiments, PEG is conjugated to block copolymer ends groups, orto one or more pendant modifiable group present in polymeric compound,such as conjugated to modifiable groups within a hydrophilic segment orblock (e.g., a second block) of a polymer (e.g., block copolymer). As anexample, a block of a copolymer can be or can be conjugated to ashielding polymer having a repeat unit of Formula V

-   -   where R¹ and R² are each independently selected from the group        consisting of hydrogen, halogen, hydroxyl, and optionally        substituted C₁-C₃ alkyl, and having a molecular weight ranging        from about 1,500 to about 15,000.

In another general approach, a monomeric residue is derived from apolymerizable monomer comprising a PEG oligomer; for example, suchmonomeric residues can be incorporated into the polymer or into one ormore blocks of a block copolymer during polymerization. In preferredembodiments, monomeric residues can be derived from a polymerizablemonomer having a pendant group comprising an oligomer of formula I

-   -   where R¹ and R² are each independently selected from the group        consisting of hydrogen, halogen, hydroxyl, and optionally        substituted C₁-C₃ alkyl, and n is an integer ranging from 2 to        20.

Polymerization

Generally, the various polymers (or polymer chains included asconstituent moieties such as blocks of a block copolymer) of thecompounds of the invention, can be prepared in any suitable manner.Suitable synthetic methods used to produce the polymers provided hereininclude, by way of non-limiting example, cationic, anionic and freeradical polymerization.

Preferably the polymers as described above are prepared by the means ofa free radical polymerization. When a free radical polymerizationprocess is used, (i) the monomer, (ii) optionally desired co-monomer(s),and (iii) an optional source of free radicals are provided to trigger afree radical polymerization process. In some embodiments, the source offree radicals is optional because some monomers may self-initiate uponheating at high temperature, or photo-activated. In certain instances,after forming the polymerization mixture, the mixture is subjected topolymerization conditions. Polymerization conditions are conditionsunder which at least one monomer forms at least one polymer, asdiscussed herein. Such conditions are optionally varied to suitablelevels and include, by way of non-limiting example, temperature,pressure, atmosphere, ratios of starting components used in thepolymerization mixture and reaction time. The polymerization isperformed neat or in any suitable solvent, and can be carried out in anysuitable manner, including, e.g., in solution, dispersion, suspension,emulsion or bulk.

In some embodiments, initiators are present in the reaction mixture. Anysuitable initiator is optionally utilized if useful in thepolymerization processes described herein. Such initiators include, byway of non-limiting example, one or more of alkyl peroxides, substitutedalkyl peroxides, aryl peroxides, substituted aryl peroxides, acylperoxides, alkyl hydroperoxides, substituted alkyl hydroperoxides, arylhydroperoxides, substituted aryl hydroperoxides, heteroalkyl peroxides,substituted heteroalkyl peroxides, heteroalkyl hydroperoxides,substituted heteroalkyl hydroperoxides, heteroaryl peroxides,substituted heteroaryl peroxides, heteroaryl hydroperoxides, substitutedheteroaryl hydroperoxides, alkyl peresters, substituted alkyl peresters,aryl peresters, substituted aryl peresters, or azo compounds. Inspecific embodiments, benzoylperoxide (BPO) and/or AIBN are used asinitiators.

In some embodiments, polymerization is effected using a controlled(living) radical polymerization process. In preferred embodiments,reversible addition-fragmentation chain transfer (RAFT) approaches areused in synthesizing polymers from ethylenic monomers. RAFT comprises afree radical degenerative chain transfer process. In some embodiments,RAFT procedures for preparing a polymer described herein employs a chaintransfer agent (CTA).

Generally, polymers or polymer chains (e.g., polymer blocks) can beindependently derived in a method comprising polymerizing in thepresence of a reversible addition-fragmentation chain-transfer (RAFT)agent. Such RAFT agents can generally have the formula Y—RL, where RL isa leaving group, typically coupled to a chain-transfer moiety, Y,through a relatively weak covalent bond. Typically, Y can form a radicalintermediate moiety, —Y.—, generated from or in the presence of aradical moiety (e.g., such as an initiator radical under initiationreaction conditions, or such as a propagating polymer chain radical, Pn,under radical polymerization conditions).

In generally preferred embodiments, the chain transfer agent (CTA) cancomprise a thiocarbonylthio moiety. For example, the CTA can comprise athiocarbonylthio moiety, —SC(═S)—, covalently bonded to an activatinggroup, Z, and to a leaving group, —RL. Such CTA can be represented forexample, by a compound having the formula RLSC(═S)Z. Various such RAFTchain-transfer agents are known for use in controlled (living) radicalpolymerizations, including various xanthates, dithiocarbamates,diothioesters and trithiocarbonates.). See for example, Moad et al., TheChemistry of Radical Polymerization, 2d Ed., Tables 9.10 to 9.18 at pp.508 to 514, Elsevier (2006), which is incorporated herein by reference.In many embodiments, the chain transfer agent (CTA) can be amacromolecular chain transfer agent (macro-CTA). For example, achain-transfer moiety, Y, of a RAFT chain transfer agent can beincorporated onto the ω-end of a polymer chain, Pn, to form a macro-CTAcomprising a polymer compound, and represented by a formula Pn-Y. (Insuch case, the polymer chain, Pn, can effectively function as a leavinggroup, RL, of the macromolecular chain transfer agent.). As incorporatedinto a compound of the invention, —Y, is referred to as a chain transferresidue. Hence, in the context of compounds of the invention derivedfrom radical polymerization, —Y can be a chain-transfer residue. Thechain transfer residue can be derived from controlled (living) radicalpolymerization of under chain polymerization conditions. Such controlledradical polymerization reactions can be effected for example in thepresence of a chain transfer agent (CTA) such as a RAFT agent (e.g.,Y-RL) or such as a macro-CTA (e.g., Pn-Y). The chain-transfer residue,—Y, is typically covalently bonded to a polymer on the ω-end thereof(also referred to as the living end of the chain extension moiety whenincluded in a macro CTA). The chain transfer residue, —Y, can preferablybe a thiocarbonylthio moiety having a formula —SC(═S)Z, where Z is anactivating group.

Various approaches are known for cleaving and/or derivatizing the chaintransfer residue, Y, to form a chain transfer residue derivative. Seefor example, Moad et al., The Chemistry of Radical Polymerization, 2dEd., pp. 538 to 539, Elsevier (2006), which is incorporated herein byreference. See also U.S. Pat. No. 6,619,409 to Charmot et al., whichdiscloses cleavage of the thiocarbonylthio control transfer agent.Derivatized chain transfer residues, can be used for effectivelycoupling one or more biomolecular agents (such as one or more affinityreagents) to the polymer, optionally through a linking moiety.

Although RAFT agents are preferably employed, other controlled (living)radical polymerization methods are also suitable in connection with theinvention. See for example, Moad et al., The Chemistry of RadicalPolymerization, Elsevier (2006), which is incorporated herein byreference. In particular, atom transfer radical polymerization (ATRP)and stable free radical polymerization (SFRP) approaches are suitable.See Moad et al., Id.

Generally, polymers can have a low polydispersity index (PDI) ordifferences in chain length. Polydispersity index (PDI) can bedetermined in any suitable manner, e.g., by dividing the weight averagemolecular weight of the polymers by their number average molecularweight. Polydispersity values approaching one are achievable usingradical living polymerization. Methods of determining molecular weightand polydispersity, such as, but not limited to, size exclusionchromatography, dynamic light scattering, matrix-assisted laserdesorption/ionization chromatography and electrospray masschromatography are well known in the art. In some embodiments, blockcopolymers of the polymeric compounds provided herein have apolydispersity index (PDI) of less than 2.0, or less than 1.5, or lessthan 1.4, or less than 1.3, or less than 1.2

Generally, polymerization processes described herein optionally occur inany suitable solvent or mixture thereof. Suitable solvents includewater, alcohol(e.g., methanol, ethanol, n-propanol, isopropanol,butanol), tetrahydrofuran (THF) dimethyl sulfoxide (DMSO),dimethylformamide (DMF), acetone, acetonitrile, hexamethylphosphoramide,acetic acid, formic acid, hexane, cyclohexane, benzene, toluene,dioxane, methylene chloride, ether (e.g., diethyl ether), chloroform,and ethyl acetate. In one aspect, the solvent includes water, andmixtures of water and water-miscible organic solvents such as DMF.

Generally, polymerization processes described herein can be effected attemperature effective for the polymerization reaction. Temperatures canbe varied based on and in consideration of other reaction aspects,including for example selections as to solvent, monomer (or comonomers)being polymerized (or copolymerized), chain transfer agent, heattransfer (exotherm control), reaction kinetics, and reactionthermodynamics. Typical temperature ranges can generally include atemperature ranging from about 2° C. to about 200° C., preferably fromabout 20° C. to about 110° C., and in some embodiments from about 40° C.to about 90° C., and or from about 50° C. to about 80° C.

Generally, polymerization processes described herein can be effected ata pressure effective for the polymerization reaction. Generally,reaction pressure is not narrowly critical, and can be at ambientpressure of about 1 atm or at higher pressures (e.g., ranging from 1 atmto about 10 atm) or a lower pressure (e.g., below 1 atm).

Generally, polymerization processes described herein can be effectedunder a reaction atmosphere effective for the polymerization reaction.For example, polymerization can be effected under an inert gasatmosphere (e.g., Ar, N2), or under ambient atmosphere.

Generally, polymerization processes described herein can be effected atvarious molar ratios of chain transfer agent (living chain transfermoieties or groups) to monomer effective for the polymerizationreaction. For example, polymerization can be effected with a molar ratioof chain transfer agent (groups) to monomer ranging from about 1:1 toabout 1:10,000, preferably from about 1:5 to about 1:5000, and mostpreferably from about 1:10 to about 1:2000In some embodiments, sch molarratio can range from about 1:10 to about 1:1500.

Generally, polymerization processes described herein can be effected atconcentrations of monomer(s) in the solvent ranging from about 5% toabout 95% by weight, preferably from about 10% to about 90% solids, byweight, and in some embodiments, from about 20% to about 80% solids, byweight, in each case relative to total weight of solution.

Generally, polymerization processes described herein can be effected atvarious molar ratios of chain transfer agent (living chain transfermoieties or groups) to initiator effective for the polymerizationreaction. For example, polymerization can be effected with a molar ratioof chain transfer agent (groups) to initiator ranging from about 1:2 toabout 50:1, and preferably from about 1:1 to about 40:1, and in someembodiments from about 2:1 to about 30:1.

Generally, polymerization processes described herein can be effected forvarious reaction times effective for the polymerization reaction. Forexample, the polymerization can be effected over a reaction time periodranging from about 0.5 hr to about 96 hr, preferably from about 1 hourto about 72 hours, more preferably from about 1 hour to 36 hours, and insome embodiments from about 2 hours to 24 hours, or from about 3 hoursto about 12 hours.

Generally, the aforementioned aspects and other factors known in the artcan be used to effect the polymerization reaction of interest. Seegenerally, for example, Moad et al., The Chemistry of RadicalPolymerization, 2d Ed., Elsevier (2006), which is incorporated herewithin this regard.

The polymer may be chemically conjugated to the bispecific affinityreagent by any standard chemical conjugation technique. The covalentbond between the polymer and the bispecific affinity reagent may benon-cleavable, or cleavable bonds may be used. Particularly preferredcleavable bonds are disulfide bonds that dissociate in the reducingenvironment of the cytoplasm. Covalent association is achieved throughchemical conjugation methods, including but not limited toamine-carboxyl linkers, amine-sulfhydryl linkers, amine-carbohydratelinkers, amine-hydroxyl linkers, amine-amine linkers,carboxyl-sulfhydryl linkers, carboxyl-carbohydrate linkers,carboxyl-hydroxyl linkers, carboxyl-carboxyl linkers,sulfhydryl-carbohydrate linkers, sulfhydryl-hydroxyl linkers,sulfhydryl-sulfhydryl linkers, carbohydrate-hydroxyl linkers,carbohydrate-carbohydrate linkers, and hydroxyl-hydroxyl linkers.Conjugation can also be performed with pH-sensitive bonds and linkers,including, but not limited to, hydrozone and acetal linkages. A largevariety of conjugation chemistries are established in the art (see, forexample, Bioconjugation, Aslam and Dent, Eds, Macmillan, 1998 andchapters therein). Alternatively, conjugation can be achieved usingstandard coordination chemistry, such as by way of a platinum complexthat forms coordinate bonds with proteinaceous entities. See, e.g., U.S.Pat. No. 5,985,566.

The bispecific polymer delivery vehicle can be used to deliver itssecond affinity reagent into cells, either for in vitro use, as aresearch reagent, or in mice or other small animal as a researchreagent. Such reagents would be useful in target validation, to showthat inhibition of a particular target has particularly favorableefficacy in cells, or to characterize and study side-effect or toxicityprofiles that result from inhibition of chosen targets. The bispecificpolymer delivery vehicle compositions may also be used as therapeutics,to inhibit the action, or to stimulate the action, of specific proteinsin humans and thereby achieve a desirable therapeutic outcome toinfluence a disease state. In addition, the compositions of the presentinvention may be used as diagnostics for the assessment of diseasestates in humans.

Bispecific polymer delivery vehicles may be formulated in any one of avariety of excipients and delivered by any route, including but notlimited to intravenous, intratumoral, intraocular, by inhalation,orally, subcutaneously, intracranially, or by a catheter, pump or otherdevice.

Cellular signaling proteins form the basis of cell behavior, and theability to intervene in selected signaling pathways would be desirableto treat a variety of disease states. Diseases that may be influenced bythe compositions and methods of the present invention include but arenot limited to cancer, either via targeting the cancer cells themselvesor targeting endothelial cells to inhibit tumor neovascularization orthrough targeting tumor stromal cells; autoimmune diseases;inflammation; wound healing; atherosclerosis; osteoarthritis; CNSdisorders; and metabolic disorders such as diabetes and obesity.

In some embodiments, polymeric carriers of the present invention havesuperior commercial viability relative to other technologies fordelivering polynucleotides, including but not limited to: decreasedimmunogenicity of the carrier following repeat in vivo administration;fewer steps needed to assemble the multiple elements of the deliveryvehicle, resulting in lower cost of goods; and reproducibility ofmanufacture, as judged by the ability to manufacture repeated batches ofproduct with less than 5%, 10%, or 20% variability in biophysical assayresults (such as GPC, DLS, TEM) between batches.

Abbreviations

Throughout the description of the present invention, various knownacronyms and abbreviations are used to describe monomers or monomericresidues derived from polymerization of such monomers. Withoutlimitation, unless otherwise noted: “BMA” (or the letter “B” asequivalent shorthand notation) represents butyl methacrylate ormonomeric residue derived therefrom; “DMAEMA” (or the letter “D” asequivalent shorthand notation) represents N,N-dimethylaminoethylmethacrylate or monomeric residue derived therefrom; “Gal” refers togalactose or a galactose residue, optionally includinghydroxyl-protecting moieties (e.g., acetyl) or to a pegylated derivativethereof (as described below); HPMA represents 2-hydroxypropylmethacrylate or monomeric residue derived therefrom; “MAA” representsmethylacrylic acid or monomeric residue derived therefrom; “MAA(NHS)”represents N-hydroxyl-succinimide ester of methacrylic acid or monomericresidue derived therefrom; “PAA” (or the letter “P” as equivalentshorthand notation) represents 2-propylacrylic acid or monomeric residuederived therefrom, “PEGMA” refers to the pegylated methacrylic monomer,CH₃O(CH₂O)₇₋₈OC(O)C(CH₃)CH₂ or monomeric residue derived therefrom. Ineach case, any such designation indicates the monomer (including allsalts, or ionic analogs thereof), or a monomeric residue derived frompolymerization of the monomer (including all salts or ionic analogsthereof), and the specific indicated form is evident by context to aperson of skill in the art.

The following Examples are for illustration purposes and are not to beconstrued as limiting the invention

EXAMPLE 1 Construction of a Ras Bispecific Polymer Delivery Vehicle

The first affinity reagent, OKT 9, is a monoclonal antibody against thehuman type 1 transferrin receptor and is available from ATCC(CRL 8021C). The hybridoma is cultured, expanded and the secreted monoclonalantibody harvested and purified using protein A or G chromatography(Antibody Engineering: A Practical Approach, McCafferty, Hoogenboom andChiswell Eds, IRL Press 1996).

The second affinity reagent, Y13-259, is a monoclonal antibody againstthe ras protein (Furth et al., 1982) and is obtained as a hybridoma fromATCC(CRL 1742). The hybridoma is cultured, expanded and the secretedmonoclonal antibody harvested and purified using protein A or Gchromatography (Antibody Engineering: A Practical Approach, McCafferty,Hoogenboom and Chiswell Eds, IRL Press 1996).

The first and second affinity reagents are co-conjugated topoly(propylacrylic acid) (PAA)-co-pyridyl disulfide acrylate (PDSA),p(PAA-co-PDSA), via disulfide exchange between the PDSA component of thepolymer and free thiols introduced onto the antibody by reaction withTraut's reagent (2-iminothiolane, Pierce Biotechnology, Rockford, Ill.).10 mg of antibody is mixed with a 10× molar excess of Traut's reagent inconjugation buffer (0.1 M phosphate buffer, pH 7.8, 0.15 M NaCl, 5 mMEDTA) for 1 hour at room temperature. The reaction mixture is purifiedusing a PD-10 desalting column containing Sephadex G-25 (MWCO 5 kD, GEHealthcare, Piscataway, N.J.) and the degree of modification isestimated by Ellman's assay (Pierce Biotechnology, Rockford, Ill.). Forthis assay, 2.5× molar excess of p(PAA-co-PDSA) is immediately added tothe modified protein and allowed to react 2 hours at room temperature inconjugation buffer. The degree of conjugation is estimated by measuringthe absorbance at 343 nm (A₃₄₃) of the pyridine-2-thione group releasedfrom PDSA upon disulfide exchange. After conjugation, the conjugate ispurified using gel permeation chromatography (GPC).

For the synthesis of p(PAA-co-PDSA), 0.007 mol PAA (Gateway ChemicalTechnology, St. Louis, Mo.), 0.00011 mol PDSA, and 0.000056 molfree-radical initiator azobisisobutyronitrile (AIBN, purified byrecrystallization from methanol) are combined in a 5 ml flask anddegassed by 4 rounds of freeze-vacuum-thaw then reacted at 60° C. for 24hours. The polymer is dissolved in 3 ml dimethyl formamide (DMF) andpurified by 3 rounds of precipitation in 500 ml diethyl ether. Polymercompositions are determined by H¹-NMR using a Bruker AVance 300 MHzinstrument and deuterated dimethyl sulfoxide (DMSO-d6, Fisher Chemical,Pittsburgh, Pa.). PDSA content is determined both by NMR and by theabsorbance at 343 nm of pyridine-2-thione released from the polymerfollowing reduction with excess of dithiothreitol (DTT). The molecularweight distribution is determined by GPC (Viscotek VE2001 sample module,VE3580 RI Detector, Waters Corp. ultrahydrogel columns) in 0.1 M sodiumphosphate buffer, pH 8 using poly(ethylene oxide) (PEO) standards(Polysciences, Inc., Warrington, Pa.).

EXAMPLE 2 Demonstration of Activity of a Ras Bispecific Polymer DeliveryVehicle

The HT1080 cell line (available from ATCC), harboring an activated N-rasgene, is cultured in DMEM with 10% FCS and antibiotics at 37 degreescentigrade in a humidified atmosphere of 10% carbon dioxide in air.Cells are expanded, plated at a density of 100,000 cells per 60 mmplastic cell culture Petri dish in 5 ml liquid cultures, incubated for15-18 h, then exposed to 0, 1, 10, 50 and 200 ug/ml of the bispecificpolymer delivery vehicle for 8 h. Cells are then removed from theplastic with trypsin/EDTA, centrifuged, resuspended in culture media,counted using a hemacytometer, and seeded at a density of 20,000cells/ml in culture media as above, but containing the sameconcentrations of the bispecific polymer delivery vehicle and 0.3% agarin 60 mm Petri dishes (5 ml/dish).

Cell cultures are incubated for 14 days at 37 degrees centigrade in ahumidified atmosphere of 10% carbon dioxide in air. The cultures arethen scored by counting the number of agar colonies per plate, incomparison to control cultures.

EXAMPLE 3 Construction of a p53 Bispecific Polymer Delivery Vehicle

The first affinity reagent is the OKT 9 antibody described above inExample 1. The second affinity reagent, PAb421, is a monoclonal antibody(Harlow et al, 1981) directed against the carboxyl terminus of human p53protein, and can rescue the transcriptional defects of the p53 mutantpresent in SW480 colorectal carcinoma cells (Selivanova et al., 1997).PAb421 is obtained as a hybridoma. The hybridomas expressing the firstand second affinity reagents are cultured, expanded and the secretedmonoclonal antibody harvested and purified using a protein A or G columnas described above in Example 1.

The first and second affinity reagents are then co-conjugated to thep(PAA-co-PDSA) polymer and purified as described above in Example 1.

EXAMPLE 4 Demonstration of Activity of p53 Bispecific Polymer DeliveryVehicle

SW480 cells are obtained from ATTC (CCL-228). Cells are grown in DMEMwith 10% FCS and antibiotics at 37 degrees centigrade in a humidifiedatmosphere of 10% carbon dioxide in air. Cells are expanded tonear-confluency, plated at a density of 500,000 cells per 100 mm plasticcell culture Petri dish in 15 ml liquid cultures, incubated for 15-18 h,then transfected using LipofectAMINE (Invitrogen, Carlsbad, Calif.)according to the manufacturers recommendations with 2 ug of theexpression plasmid PG-13 CAT (Kern et al., 1992). Cells are incubatedand exposed to 0, 1, 10, 50 and 200 ug/ml of the bispecific polymerdelivery vehicles in liquid culture for 48 h, then harvested and assayedfor chloramphenicol acetyltransferase (CAT) activity. Cell lysates areprepared by 3 cycles of freeze-thawing, and the cleared supernatant isassayed for CAT activity using a standard 14-C chloramphenicolthin-layer chromatography assay, in comparison to control cultures.

EXAMPLE 5 Creation and Demonstration of Activity of a Ras BispecificPolymer Delivery Vehicle Using a Bispecific Fusion Protein

The bispecific affinity reagent in this example is a bispecificantibody. The first and second affinity reagents are assembled fromrecombinant DNA clones by standard recombinant and PCR techniques. Thefirst affinity reagent is derived from the monoclonal antibody hybridomaOKT 9 directed against the transferrin receptor, and the second affinityreagent is derived from the ras antibody from the hybridoma Y13-259,both described above in Example 1. mRNA is isolated from the respectivehybridoma cells, reverse-transcribed into cDNA using antisense oligo-dTor gene-specific primers, and cloned into a plasmid vector. Clones aresequenced and characterized. They are then engineered according tostandard protocols to combine the heavy and light chains of eachantibody, separated by a short peptide linker, into a bacterial ormammalian expression vector as previously described, and expressed andpurified according to well-established protocols in mammalian cells(Kufer et al., 2004; Antibody Engineering: A Practical Approach,McCafferty, Hoogenboom and Chiswell Eds, IRL Press 1996). The resultingbispecific affinity reagent thus binds to the transferrin receptor cellsurface antigen, and to the ras intracellular target.

The bispecific scFv fragment is then cloned into a mammalian expressionvector, and the vector transfected into CHO cells. The CHO cells arescreened for productivity of the scFv, and the resulting cells used toexpress the bispecific scFv, which is then harvested and purified bystandard chromatographic techniques (see Antibody Engineering: APractical Approach, McCafferty, Hoogenboom and Chiswell Eds, IRL Press1996).

The purified bispecific scFv is then conjugated to the p(PAA-co-PDSA)polymer and purified as described above in Example 1, and assayed foractivity as described above in Example 2.

EXAMPLE 6 Creation and Demonstration of Activity of a p53 BispecificPolymer Delivery Vehicle Using a Bispecific Fusion Protein

The bispecific affinity reagent in this example is a bispecificantibody. The first and second affinity reagents are assembled fromrecombinant DNA clones by standard recombinant and PCR techniques. Thefirst affinity reagent is derived from the monoclonal antibody hybridomaOKT 9 directed against the transferrin receptor, and the second affinityreagent is derived from the p53 antibody from the hybridoma PAb421, bothdescribed above in Examples 1 and 3. mRNA is isolated from therespective hybridoma cells, reverse-transcribed into cDNA usingantisense oligo-dT or gene-specific primers, and cloned into a plasmidvector. Clones are sequenced and characterized. They are then engineeredaccording to standard protocols to combine the heavy and light chains ofeach antibody, separated by a short peptide linker, into a bacterial ormammalian expression vector as previously described, and expressed andpurified according to well-established protocols in mammalian cells(Kufer et al., 2004; Antibody Engineering: A Practical Approach,McCafferty, Hoogenboom and Chiswell Eds, IRL Press 1996). The resultingbispecific affinity reagent thus binds to the transferrin receptor cellsurface antigen, and to the p53 intracellular target.

The bispecific scFv fragment is then cloned into a mammalian expressionvector, and the vector transfected into CHO cells. The CHO cells arescreened for productivity of the scFv, and the resulting cells used toexpress the bispecific scFv, which is then harvested and purified bystandard chromatographic techniques (see Antibody Engineering: APractical Approach, McCafferty, Hoogenboom and Chiswell Eds, IRL Press1996).

The purified bispecific scFv is then conjugated to the p(PAA-co-PDSA)polymer and purified as described above in Example 1, and assayed foractivity as described above in Example 4.

In the following examples, a novel diblock copolymer carrier isdescribed that facilitates intracellular delivery of a bispecificpeptide containing two functional domains, connected by directpolymer-peptide conjugation: (1) a cell binding and endocytotic updatedomain (the protein transduction domain, penetratin) and (2) apro-apoptotic Bak-BH3 peptide with pharmaceutical properties. Thepolymer was prepared using reversible addition fragmentation chaintransfer (RAFT) (Chiefari et al. Macromolecules. 1998; 31(16):5559-5562)to form an end-functionalized, modular diblock copolymer thatincorporates an N-(2-hydroxypropyl) methacrylamide (HPMA) first block toenhance water solubility and favorable pharmacokinetic properties and apH-responsive polymer block composed of dimethylaminoethyl methacrylate(DMAEMA), propylacrylic acid (PAA), and butyl methacrylate (BMA) thatprovides a mechanism for peptide endosomal escape. This polymericcarrier was conjugated to the bi-specific peptide via a reducibledisulfide bond that ensures polymer release of the peptide upon deliveryto the cytosol. The polymer exhibits pH-dependent membrane destabilizingbehavior, and peptide-polymer conjugation was shown to significantlyenhance peptide intracellular delivery and downstream pro-apoptoticbioactivity. The results show that this multifunctional diblock polymercarrier demonstrates the delivery of a peptide drug to an intracellulartarget.

EXAMPLE 7 Functional Design of Poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)]

FIG. 1 shows the polymer design for Poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)].Multifunctional properties were incorporated via RAFT polymer synthesisstrategies using a pyridyl disulfide end-functionalized CTA to form adiblock architecture designed to possess aqueous solubility andpH-dependent membrane destabilizing properties. The monomer chemicalfunctionalities highlighted in FIG. 1 were chosen in order to producethe desired properties for each polymer block. Importantly, module 3 wasdesigned to be near charge neutrality at physiologic pH (approximately50% DMAEMA protonation and 50% PAA deprotonation predicted) and toundergo a transition to a more hydrophobic and positively charged statein lower pH environments.

EXAMPLE 8 Preparation of Thiol Reactive Polymer: Synthesis ofTrithiocarbonic acid1-cyano-1-methyl-3-[2-(pyridin-2-yldisulfanyl)-ethylcarbamoyl]-propylester ethyl ester (PyrECT)

The 4-cyano-4-(ethylsulfanylthiocarbonyl)sulfanylvpentanoic acid (ECT)precursor was synthesized as shown in FIG. 2. The pyridyl disulfidefunctionalized RAFT chain transfer agent (CTA) was synthesized by firstconverting ECT to the NHS ester followed by reaction withpyridyldithio-ethylamine.

ECT (1.05 g, 4 mmol) and N-hydroxysuccinimide (0.460 g, 4 mmol) weredissolved in 100 mL of chloroform. The mixture was then cooled to 0° C.at which time N,N′-dicyclohexylcarbodiimide (0.865 mg, 4.2 mmol) wasadded. The solution was maintained at 0° C. for 1 hour and then allowedto react at room temperature for 22 hours. The solution was thenfiltered to remove the dicyclohexyl urea and the solution concentratedvia rotary evaporation. The resultant solid was then dried under vacuumand used without any further purification. NHS ECT (1.80 g, 5.0 mmol)and pyridyldithio-ethylamine (0.90 g, 5.0 mmol) where then separatelydissolved in 200 and 300 mL of chloroform, respectively. The solution ofpyridyldithio-ethylamine was then added dropwise as three fractions 20minutes apart. The mixture was then allowed to react at room temperaturefor 2 hours. After solvent removal, two successive columnchromatographies (Silica gel 60, Merk) were performed (ethylacetate:hexane 50:50; ethyl acetate:hexane 70:30 v/v) yielding a viscousorange solid. 1H NMR 200 MHz (CDCl3, RT, ppm) 1.29-1.41 [t, CH3CH2S:3H], 1.85-1.93 [s, (CH₃)C(CN): 3H], 2.33-2.59 [m, C(CH₃)(CN)(CH₂CH₂):4H], 2.86-2.97 [t, CH₂SS: 2H], 3.50-3.61 [t, NHCH₂: 2H], 7.11-7.22 [m,Ar Para CH: 1H], 7.46-7.52 [m, Ar CH Ortho: 1H], 7.53-7.62 [br, NH: 1H],7.53-7.68 [m, Ar meta CH: 1H], 8.47-8.60 [m, meta CHN, 1H].

Preparation of Thiol Reactive Polymer: Raft Polymerization of PyridylDisulfide Functionalized poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)]

The RAFT polymerization of N-(2-hydroxypropyl) methacrylamide (HPMA) wasconducted in methanol (50 weight percent monomer:solvent) at 70° C.under a nitrogen atmosphere for 8 hours using2,2′-azo-bis-isobutyrylnitrile (AIBN) as the free radical initiator. Themolar ratio of CTA to AIBN was 10 to 1 and the monomer to CTA ratio wasset so that a molecular weight of 25,000 g/mol would be achieved if at100% conversion. The poly(HPMA) macro-CTA was isolated by repeatedprecipitation into diethyl ether from methanol.

The macro-CTA was dried under vacuum for 24 hours and then used forblock copolymerization of dimethylaminoethyl methacrylate (DMAEMA),propylacrylic acid (PAA), and butyl methacrylate (BMA). Equimolarquantities of DMAEMA, PAA, and BMA ([M]_(o)/[CTA]_(o)=250) were added tothe HPMA macroCTA dissolved in N,N-dimethylformamide (25 wt % monomerand macroCTA to solvent). The radical initiator V70 was added with a CTAto initiator ratio of 10 to 1. The polymerization was allowed to proceedunder a nitrogen atmosphere for 18 hours at 30° C. Afterwards, theresultant diblock polymer was isolated by precipitation 4 times into50:50 diethyl ether/pentane, redissolving in ethanol betweenprecipitations. The product was then washed 1 time with diethyl etherand dried overnight in vacuo.

Gel permeation chromatography (GPC) was used to determine molecularweight and polydispersity (Mw/Mn, PDI) of both the poly(HPMA) macroCTAand the diblock copolymer in DMF. Molecular weight calculations werebased on column elution times relative to polymethyl methacrylatestandards using HPLC-grade DMF containing 0.1 wt % LiBr at 60° C. as themobile phase. Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) wasused to reduce the polymer end pyridyl disulfide, releasing2-pyridinethione. Based on the experimentally determined polymermolecular weight and the molar extinction coefficient of2-pyridinethione at 343 nm (8080 M⁻¹cm⁻¹) in aqueous solvents, percentend group preservation was determined for the poly(HPMA) macroCTA andthe diblock copolymer.

EXAMPLE 9 Polymer-Peptide Conjugation

Fusion with the peptide transduction domain peptide transporting (alsoknow as the Antennapedia peptide (Antp) sequence was utilized tosynthesize a cell internalizing form of the Bak-BH3 peptide (Antp-BH3)containing a carboxy-terminal cysteine residue(NH2-RQIKIWFQNRRMKWKKMGQVGRQLAIIGDDINRRYDSC—COOH). To ensure free thiolsfor conjugation, the peptide was reconstituted in water and treated for1 hour with the disulfide reducing agent TCEP immobilized within anagarose gel. The reduced peptide (400 μM) was then reacted for 24 hourswith the pyridyl disulfide end-functionalized polymer in phosphatebuffer (pH 7) containing 5 mM ethylenediaminetetraacetic acid (EDTA).

As shown in FIG. 3, reaction of the pyridyl disulfide polymer end groupwith the peptide cysteine creates 2-pyridinethione, which can bespectrophotometrically measured to characterize conjugation efficiency.To further validate disulfide exchange, the conjugates were run on anSDS-PAGE 16.5% tricine gel. In parallel, aliquots of the conjugationreactions were treated with immobilized TCEP prior to SDS-PAGE to verifyrelease of the peptide from the polymer in a reducing environment.

Conjugation reactions were conducted at polymer/peptide stoichiometriesof 1, 2, and 5. UV spectrophotometric absorbance measurements at 343 nmfor 2-pyridinethione release indicated conjugation efficiencies of 40%,75%, and 80%, respectively (moles 2-pyridinethione/moles peptide). AnSDS PAGE gel was utilized to further characterize peptide-polymerconjugates. At a polymer/peptide molar ratio of 1, a detectable quantityof the peptide formed dimers via disulfide bridging through the terminalcysteine. However, the thiol reaction to the pyridyl disulfide wasfavored, and the free peptide band was no longer visible atpolymer/peptide ratios equal to or greater than 2 (FIG. 4A). By treatingthe conjugates with the reducing agent TCEP, it was possible to cleavethe polymer-peptide disulfide linkages as indicated by the appearance ofthe peptide band in these samples (FIG. 4B).

EXAMPLE 10 pH-dependent Membrane Destabilizing Properties ofpoly[HPMA]-b-[(PAA)(BMA)(DMAEMA)]

In order to assess the polymer's potential for endosomolytic activity, amembrane disruption assay was utilized to measure the capacity of thepolymer to trigger pH-dependent disruption of lipid bilayer membranes asshown in FIG. 5. Whole human blood was drawn and centrifuged for plasmaremoval. The remaining erythrocytes were washed three times with 150 mMNaCl and resuspended into phosphate buffers corresponding tophysiological (pH 7.4), early endosome (pH 6.6), and late endosome (pH5.8) environments. The polymer (1-40 μg/mL) or 1% Triton X-100 was addedto the erythrocyte suspensions and incubated for 1 hour at 37° C. Intacterythrocytes were pelleted via centrifugation, and the hemoglobincontent within the supernatant was measured via absorbance at 541 nm.Percent hemolysis was determined relative to Triton X-100. Polymerhemolysis was quantified at concentrations ranging from 1-40 μg/mLrelative to 1% v/v Triton X-100. This experiment was completed 2 timesin triplicate, yielding similar results. The data shown represent asingle experiment conducted in triplicate±standard deviation.

Red blood cell hemolysis measures pH-dependent membrane disruptionproperties of the diblock copolymer at pH values mimicking physiologic(7.4), early endosomal (6.6) and late endosomal (5.8) environments. Atphysiologic pH, no significant red blood cell membrane disruption wasobserved even at polymer concentrations as high as 40 μg/mL (FIG. 5).However, as the pH was lowered to endosomal values, a significantincrease in hemolysis was detected, with greater membrane disruption atpH 5.8 compared to 6.6. The hemolytic behavior of the polymer correlatedto polymer concentration, with nearly 70% erythrocyte lysis occurring at40 μg/mL polymer in pH 5.8 buffer. This sharp “switch” to a membranedestabilizing conformation at endosomal pH combined with negligiblemembrane activity in the physiologic pH range indicates potential forthis polymer as a non-toxic intracellular delivery vehicle.

EXAMPLE 11 Characterization of Intracellular Delivery in HeLa Cells

HeLas, human cervical carcinoma cells (ATCC CCL-2), were maintained inminimum essential media (MEM) containing L-glutamine, 1%penicillin-streptomycin, and 10% FBS. Prior to experiments, HeLas wereallowed to adhere overnight in 8-well chamber slides (20,000 cells/well)for microscopy or 96-well plates (10,000 cells/well) for other assays.Polymer-peptide conjugates and controls were added in MEM with 1% FBS.

Polymer intracellular delivery potential was evaluated followingbioconjugation to the Bak-BH3 peptide fused with the Antp (penetratin)cell penetrating peptide. BH3 fusion to Antp has been extensivelystudied as a cell translocation domain and has previously been found totrigger apoptotic signaling (Li et al. Neoplasia (New York, N.Y. 2007;9(10):801-811). However, it is believed that therapeutics delivered viapeptidic transduction domains may suffer from hindered potency due tosequestration within intracellular vesicles (Sugita et al. BritishJournal of Pharmacology. 2008; 153(6):1143-1152).

The following in vitro studies demonstrate that the combined Antp-BH3peptide cytoplasmic delivery and pro-apoptotic functionality wasenhanced by conjugation to the diblock polymer.

Microscopic Analysis of Conjugate Endosomal Escape

An amine reactive Alexa-488 succinimidyl ester was mixed at a 1 to 1molar ratio with the Antp-BH3 peptide in anhydrous dimethyl formamide(DMF). Unreacted fluorophore and organic solvent were removed using aPD10 desalting column, and the fluorescently labeled peptide waslyophilized. Alexa-488 labeled Antp-BH3 was conjugated to the polymer asdescribed above. Free peptide or polymer-peptide conjugate was appliedto HeLas grown on chambered microscope slides at a concentration of 25μM Antp-BH3. Cells were treated for 15 minutes, washed twice with PBS,and incubated in fresh media for an additional 30 minutes. The sampleswere washed again and fixed with 4% paraformaldehyde for 10 minutes at37° C. Slides were mounted with ProLong Gold Antifade reagent containingDAPI and imaged using a fluorescent microscope.

To study the effects of polymer conjugation on peptide endosomal escape,the Alexa-488 labeled peptide was analyzed by fluorescent microscopy.The fluorescently labeled peptide was delivered alone or as the polymerbioconjugate. Microscopic analysis revealed clear differences in peptideintracellular localization following polymer conjugation (FIGS. 6A and6B). The peptide alone displayed punctate staining, indicative ofendosomal compartmentalization. Samples delivered polymer-peptideconjugate exhibited a dispersed fluorescence pattern, consistent withpeptide diffusion throughout the cytoplasm. Representative imagesillustrating (FIG. 6A) punctate peptide staining in the samplesdelivered peptide alone and (FIG. 6B) dispersed peptide fluorescencewithin the cytosol following delivery of peptide-polymer conjugate.Samples were treated for 15 minutes with 25 μM peptide and prepared formicroscopic examination following DAPI nuclear staining.

Measurement of Conjugate Cytotoxicity

Bioconjugate efficacy for triggering tumor cell death was determinedusing a lactate dehydrogenase (LDH) cytotoxicity assay. At the end ofeach time point, cells were washed two times with PBS and then lysedwith cell lysis buffer (100 μL/well, 20 mM Tris-HCl, pH 7.5, 150 mMNaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1mM 13-glycerophosphate, 1 mM sodium orthovanadate) for 1 hour at 4° C.20 μl of lysate from each sample was diluted into 80 μl PBS, and LDH wasquantified by mixing with 100 μL of the LDH substrate solution.Following a 10 minute incubation, LDH was determined by measuringabsorbance at 490 nm. Percent viability was expressed relative tosamples receiving no treatment.

To assess polymer-peptide conjugate bioactivity, a cytotoxicity studywas conducted in HeLa cervical cancer cells. The Antp-BH3 polymerconjugate was found to potently trigger HeLa cell death in a dosedependent fashion. Less than 50% HeLa viability was detected after 6hours of treatment with 10 μM peptide conjugate (FIG. 7A), and samplesreceiving 25 μM peptide conjugate showed little if any viable cellsfollowing as little as 4 hours of exposure (FIG. 7B). Control samplesreceiving peptide or polymer alone displayed negligible treatmenteffect, and there was no difference between these control treatmentgroups. Importantly, Antp-BH3 poly(HPMA) conjugates that lacked thepH-responsive block were similar to both control groups and did notresult in significant toxicity, further validating the functionality ofthe endosomolytic block.

Flow Cytometry Evaluation of Mitochondrial Membrane Potential

Loss of mitochondrial membrane potential, a known indicator forapoptosis, was assessed using the JC-1 dye. JC-1 exhibits greenfluorescence when dispersed in the cytosol, and in healthy cells, itforms red-fluorescent aggregates at the mitochondrial membrane(Cossarizza et al. Biochemical and biophysical research communications.1993; 197(1):40-45). HeLas were incubated for 2 hours with 10 μM peptideor equivalent conjugate or polymer alone. JC-1 was added at a finalconcentration of 5 μg/mL and incubated for 15 minutes. Cells were washed2 times with PBS, trypsinized, and resuspended in 0.5% BSA for flowcytometric analysis. Percent of cells displaying mitochondrialdepolarization was quantified based on the number of green fluorescentcells that were negative for red fluorescence. Here, a significant lossof red fluorescent JC-1 aggregates and therefore a loss in mitochondrialpolarization was detected following treatment with both the Antp-BH3peptide and the polymer peptide conjugate (FIG. 8A). Polymer controlswere similar to cells receiving no treatment while Antp-BH3 alone and ina polymer conjugate resulted in an approximately 4- and 10-foldincrease, respectively, in percent of cells exhibiting loss ofmitochondrial polarity.

Caspase 3/7 Activity Assay

Caspase 3/7 activation was measured using a commercially available assaykit. This assay utilizes a profluorescent caspase 3/7 substrate thatonce enzymatically cleaved becomes fluorescent allowing fordetermination of relative enzyme activity using a fluorescent platereader. Here, HeLas were incubated for 30 minutes with 25 μM peptide(alone or as polymer conjugate) in addition to polymer alone in aquantity equivalent to the conjugate samples. Afterwards, a caspase 3/7fluorigenic indicator was added directly to the culture media for eachsample. Plates were shaken for 1 hour and then assayed using afluorescent plate reader. Data were expressed as percent caspaseactivity relative to samples receiving no treatment.

Activation of caspases 3 and 7, which is indicative of pro-apoptoticsignaling, can be measured using a profluorescent substrate specific tothese proteases. FIG. 8B shows that controls containing the polymeralone displayed equivalent caspase activity relative to negativecontrols receiving no treatment. However, rapid caspase activation(approximately 2.5-fold) was detected following treatment with theAntp-BH3 peptide by itself or in the polymer conjugate form. The similareffects of Antp-BH3 alone or as a polymer conjugate could indicate thatcaspase signaling is saturated by treatment with the peptide alone orthat other positive feedback mechanisms exist for amplification ofperturbations in caspase activation state. Minimally, these resultssuggest that there was no steric hindrance or other reductions inpeptide-induced caspase activity as a result of conjugation to thepolymer.

The invention claimed is:
 1. A composition for delivering an agent to acell, comprising: a bispecific affinity reagent, comprising a firstaffinity reagent covalently linked to a second affinity reagent, whereinthe first affinity reagent binds to a molecule on the surface of a cell,and the second affinity reagent binds to an intracellular target, andwherein the bispecific affinity reagent comprises an antibody, antibodyfragment, antibody-like molecule, a single protein, or a specific scFv;and a pH-responsive, membrane destabilizing polymer.
 2. The compositionof claim 1, wherein the first affinity reagent and the second affinityreagent are included in a single polypeptide chain.
 3. A composition fordelivering an agent to a cell, comprising: a bispecific affinityreagent, comprising a first affinity reagent covalently linked to asecond affinity reagent, wherein the first affinity reagent binds to amolecule on the surface of a cell, and the second affinity reagent bindsto an intracellular target, and wherein the first affinity reagent is anantibody, antibody fragment, antibody-like molecule, a single protein,peptide, oligosaccharide, or aptamer; and a pH-responsive, membranedestabilizing polymer.
 4. The composition of claim 3 wherein the firstaffinity reagent is a small molecule selected from the group consistingof folate or a ligand for the asialoglycoprotein receptor.
 5. Thecomposition of claim 3, wherein the peptide is an RGD peptide.
 6. Thecomposition of claim 1, wherein the first affinity reagent comprises aprotein transduction domain.
 7. The composition of claim 6, wherein theprotein transduction domain comprises antennapedia, TAT, VP22,transportan, penetratin, polyarginine, fragments thereof, orcombinations thereof.
 8. A composition for delivering an agent to acell, comprising: a bispecific affinity reagent, comprising a firstaffinity reagent covalently linked to a second affinity reagent, whereinthe first affinity reagent binds to a molecule on the surface of a cell,and the second affinity reagent binds to an intracellular target, andwherein the second affinity reagent is an antibody, antibody fragment,antibody-like molecule, peptide, or aptamer; and a pH-responsive,membrane destabilizing polymer.
 9. The composition of claim 8, whereinthe peptide is BH3 or BAK-BH3.
 10. A composition for delivering an agentto a cell, comprising: a bispecific affinity reagent, comprising a firstaffinity reagent covalently linked to a second affinity reagent, whereinthe first affinity reagent binds to a molecule on the surface of a cell,and the second affinity reagent binds to an intracellular target, andwherein the intracellular target is a DNA binding protein, a cytoplasmicdomain of a membrane receptor, a kinase, a GTPase, a phosphatase, or aprotein in the Bel family of proteins; and a pH-responsive, membranedestabilizing polymer.
 11. The composition of claim 10, wherein the DNAbinding protein is p53.
 12. The composition of claim 10, wherein theGTPase is ras.
 13. The composition of claim 10, wherein the polymer is acopolymer comprising: a membrane destabilizing segment comprising aplurality of anionic species that are anionic at about neutral pH, and aplurality of hydrophobic species, and, optionally a plurality ofcationic species that are cationic at about neutral pH.
 14. Thecomposition of claim 13, wherein the copolymer further comprises: anamphiphilic or hydrophilic segment.
 15. A composition for delivering anagent to a cell, comprising: a bispecific affinity reagent, comprising afirst affinity reagent covalently linked to a second affinity reagentthrough a cleavable linker, wherein the first affinity reagent binds toa molecule on the surface of a cell, and the second affinity reagentbinds to an intracellular target, and a pH-responsive, membranedestabilizing polymer.
 16. A composition for delivering an agent to acell, comprising: a bispecific affinity reagent, comprising a firstaffinity reagent covalently linked to a second affinity reagent, whereinthe first affinity reagent binds to a molecule on the surface of a cell,and the second affinity reagent binds to an intracellular target, and apH-responsive, membrane destabilizing polymer, wherein thepH-responsive, membrane destabilizing polymer comprises a plurality ofpendant linking groups, wherein the first affinity reagent is linked tothe polymer and wherein the second affinity reagent is linked to thepolymer.
 17. The composition of claim 16, wherein the first affinityreagent is linked to an end group of the polymer.
 18. The composition ofclaim 16, wherein the second affinity reagent is linked to one of theplurality of pendant linking groups.
 19. The composition of claim 16,further comprising a plurality of second affinity reagents, wherein theplurality of second affinity reagents is linked to the polymer via thependant linking groups.
 20. The composition of claim 16, furthercomprising a plurality of first affinity reagents, wherein the pluralityof first affinity reagents is linked to the polymer via the pendantlinking groups.
 21. The composition of claim 1, further comprising atherapeutic or diagnostic agent.
 22. The composition claim 16, whereinthe second affinity reagent is cleavably linked to the polymer.
 23. Thecomposition of claim 16, wherein the second affinity reagent is anantibody, antibody fragment, antibody-like molecule, or peptide.